Electrochemical sensor using intercalative, redox-active moieties

ABSTRACT

Compositions and methods for electrochemical detection and localization of genetic point mutations, common DNA lesions and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Ser. No.09/753,362, filed Dec. 29, 2000, which is a continuation of Ser. No.09/056,995, filed Apr.8, 1998, now issued U.S. Pat. No. 6,221,586, whichclaims priority under §119(e) to U.S. Provisional application Serial No.60/043,146, filed Apr. 9, 1997, the contents of which are herebyincorporated by reference in their entirety.

GOVERNMENT RIGHTS

[0002] The U.S. Government may have certain rights in this inventionpursuant to Grant No. GM 49216 awarded by the National Instituted ofHealth.

FIELD OF THE INVENTION

[0003] The present invention relates to the detection and localizationof base-pair mismatches and other perturbations in base-stacking withinan oligonucleotide duplex.

DESCRIPTION OF RELATED ART

[0004] It is now well known that mutations in DNA can lead to severeconsequences in metabolic functions (e.g., regulation of geneexpression, modulation of protein production) which ultimately areexpressed in a variety of diseases. For example, a significant number ofhuman cancers are characterized by a single base mutation in one of thethree ras genes (Bos, 1989). In order to unravel the genetic componentsof such diseases, it is of utmost importance to develop DNA sensors thatare capable of detecting single-base mismatches rapidly and efficientlyand to establish routine screening of disease-related genetic mutationsbased on such sensors (Skogerboe, 1993; Southern, 1996; Chee, 1996; Eng,1997).

[0005] Various methods that have been developed for the detection ofdifferences between DNA sequences rely on hybridization events todifferentiate native versus mutated sequences and are limited by thesmall differences in base-pairing energies caused by point mutationswithin extended polynucleotides (Millan, 1993; Hashimoto, 1994; Xu,1995; Wang, 1996; Lockhart, 1996; Alivisatos, 1996; Korriyoussoufi,1997; Elghanian, 1997; Lin, 1997; Herne, 1997). Typically, a nucleicacid hybridization assay to determine the presence of a particularnucleotide sequence (i.e. the “target sequence”) in either RNA or DNAcomprises a multitude of steps. First, an oligonucleotide probe having anucleotide sequence complementary to at least a portion of the targetsequence is labeled with a readily detectable atom or group. When thelabeled probe is exposed to a test sample suspected of containing thetarget nucleotide sequence, under hybridizing conditions, the targetwill hybridize with the probe. The presence of the target sequence inthe sample can be determined qualitatively or quantitatively in avariety of ways, usually by separating the hybridized and non-hybridizedprobe, and then determining the amount of labeled probe which ishybridized, either by determining the presence of label in probe hybridsor by determining the quantity of label in the non-hybridized probes.Suitable labels may provide signals detectable by luminescence,radioactivity, colorimetry, x-ray diffraction or absorption, magnetismor enzymatic activity, and may include, for example, fluorophores,chromophores, radioactive isotopes, enzymes, and ligands having specificbinding partners. However, the specific labeling method chosen dependson a multitude of factors, such as ease of attachment of the label, itssensitivity and stability over time, rapid and easy detection andquantification, as well as cost and safety issues. Thus, despite theabundance of labeling techniques, the usefulness, versatility anddiagnostic value of a particular system for detecting a material ofinterest is often limited.

[0006] Some of the currently used methods of mismatch detection includesingle-strand conformation polymorphism (SSCP) (Thigpen, 1992; Orita,1989), denaturing gradient gel electrophoresis (DGGE) (Finke, 1996;Wartell, 1990; Sheffield, 1989), RNase protection assays (Peltonen andPulkkinen, 1986; Osborne, 1991), allele-specific oligonucleotides (Wu,1989), allele-specific PCR (Finke, 1996), and the use of proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991).

[0007] In the first three methods, the appearance of a newelectrophoretic band is observed by polyacrylamide gel electrophoresis.SSCP detects the differences in speed of migration of single-strandedDNA sequences in polyacrylamide gel electrophoresis under differentconditions such as changes in pH, temperature, etc. A variation in thenucleotide base sequence of single-standed DNA segments (due to mutationor polymorphism) may lead to a difference in spatial arrangement andthus in mobility. DGGE exploits differences in the stability of DNAsegments in the presence or absence of a mutation. Introduction of amutation into double-stranded sequences creates a mismatch at themutated site that destabilizes the DNA duplex. Using a gel with anincreasing gradient of formamide (denaturation gradient gel), the mutantand wild-type DNA can be differentiated by their altered migrationdistances. The basis for the RNase protection assay is that the RNase Aenzyme cleaves mRNA that is not fully hybridized with its complementarystrand, whereas a completely hybridized duplex is protected from RNase Adigestion. The presence of a mismatch results in incompletehybridization and thus cleavage by RNase A at the mutation site.Formation of these smaller fragments upon cleavage can be detected bypolyacrylamide gel electrophoresis. Techniques based on mismatchdetection are generally being used to detect point mutations in a geneor its mRNA product. While these techniques are less sensitive thansequencing, they are simpler to perform on a large number of tumorsamples. In addition to the RNase A protection assay, there are otherDNA probes that can be used to detect mismatches, through enzymatic orchemical cleavage. See, e.g., Smooker and Cotton, 1993; Cotton, 1988;Shenk, 1975. Other enzymatic methods include for example the use of DNAligase which covalently joins two adjacent oligonucleotides which arehybridized on a complementary target nucleic acid, see, for exampleLandegren (1988). The mismatch must occur at the site of ligation.

[0008] Alternatively, mismatches can also be detected by shifts in theelectrophoretic mobility of mismatched duplexes relative to matchedduplexes (Cariello, 1988). With either riboprobes or DNA probes, thecellular mRNA or DNA which may contain a mutation can be amplified usingpolymerase chain reaction (PCR) prior to hybridization. Changes in DNAof the gene itself can also be detected using Southern hybridization,especially if the changes are gross rearrangements, such as deletionsand insertions.

[0009] DNA sequences of the specified gene which have been amplified byuse of PCR may also be screened using allele-specific oligonucleotideprobes. These probes are nucleic acid oligomers, each of which iscomplementary to a corresponding segment of the investigated gene andmay or may not contain a known mutation. The assay is performed bydetecting the presence or absence of a hybridization signal for thespecific sequence. In the case of allele-specific PCR, the PCR techniqueuses unique primers which selectively hybridize at their 3′-ends to aparticular mutated sequence. If the particular mutation is not present,no amplification product is observed.

[0010] In addition, restriction fragment length polymorphism (RFLP)probes for the gene or surrounding marker genes can be used to scorealteration of an allele or an insertion in a polymorphic fragment.However, since the recognition site of restriction endonucleases rangesin general between 4 to 10 base pairs, only a small portion of thegenome is monitored by any one enzyme.

[0011] Another means for identifying base substitution is directsequencing of a nucleic acid fragment. The traditional methods are basedon preparing a mixture of randomly-termiated, differentially labeled DNAfragments by degradation at specific nucleotides, or by dideoxy chaintermination of replicating strands (Maxam & Gilbert, 1980; Sanger,1977). Resulting DNA fragments in the range of 1 to 500 basepairs arethen separated on a gel to produce a ladder of bands wherein theadjacent samples differ in length by one nucleotide. The other methodfor sequencing nucleic acids is sequencing by hybridization (SBH,Drmanac, 1993). Using mismatch discriminative hybridization of shortn-nucleotide oligomers (n-mers), lists of constitutent n-mers may bedetermined for target DNA. The DNA sequence for the target DNA may beassembled by uniquely overlapping scored oligonucleotides. Yet anotherapproach relies on hybridization to high-density arrays ofoligonucleotides to determine genetic variation. Using a two-colorlabeling scheme simultaneous comparison of a polymorphic target to areference DNA or RNA can be achieved (Lipshutz, 1995; Chee, 1996; Hacia,1996).

[0012] Each of these known prior art methods for detecting base pairmismatches has limitations that affect adequate sensitivity, specificityand ease of automation of the assay. In particular, these methods areunable to detect mismatches independent of sequence composition andrequire carefully controlled conditions, and most methods detectmultiple mismatches only. Additional shortcomings that limit thesemethods include high background signal, poor enzyme specificity, and/orcontamination.

[0013] Over the last decade, attention has also focused on DNA as amedium of charge transfer in photoinduced electron transfer reactionsand its role in mutagenesis and carcinogenesis. For example, studieswere performed using various octahedral metal complexes (which bindtightly to DNA by intercalation) as donors and acceptors forphotoinduced electron transfer. Dppz complexes of ruthenium, osmium,cobalt, nickel, and rhenium showed tight intercalative binding andunique photophysical and electrochemical properties. No photoluminesencewas observed upon irradiation of the metal complexes in aqueous solutionin absence of DNA (as a result of quenching by proton transfer from thesolvent), whereas in the presence of DNA excitation of the complexafforded significant, long-wavelength emission (because now theintercalated complex was protected from quenching). Studies usingrhodium intercalators containing phenanthrenequinone-diimine (phi)ligands displayed tight DNA binding by preferential intercalation, somewith affinities and specifities approaching DNA-binding proteins.

[0014] Photoinduced electron transfer using DNA as a molecular bridgehas been established in various systems. Using metal complexesintercalated into the base stack of DNA as donor and acceptor it hasbeen proposed that the DNA π-stack could promote electron transfer atlong range. Additionally, the products of redox-triggered reactions ofDNA bases have been detected at sites remote from intercalating oxidants(Hall, 1996; Dandliker, 1997; Hall, 1997; Arkin, 1997). For example, ithas been shown that a metallointercalator can promote oxidative DNAdamage through long-range hole migration from a remote site. OligomericDNA duplexes were prepared with a rhodium intercalator covalentlyattached to one end and separated spatially from 5′-GG-3′ doublet sitesof oxidation. Rhodium-induced photooxidation occurred specifically atthe 5′-G in the 5′-GG-3′ doublets and was observed up to 37 Å away fromthe site of rhodium intercalation.. In addition it was found thatrhodium intercalators excited with 400 nm light, initiated the repair ofa thymine dimer incorporated site-specifically in the center of asynthetic 16-mer oligonucleotide duplex. The repair mechanism wasthought to proceed via oxidation of the dimer by the intraligand excitedstate of the rhodium complex, in which an electron deficiency (hole) islocalized on the intercalated phi ligand. Like electron transfer betweenmetallointercalators, the efficiencies of long-range oxidative processeswere found to be remarkably sensitive to the coupling of the reactantsinto the base stack (Holmlin, 1997) and depended upon the integrity ofthe base stack itself (Kelley, 1997c, 1997d; Hall, 1997; Arkin, 1997) aswell as on the oxidation potential. Perturbations caused by mismatchesor bulges greatly diminished the yields of DNA-mediated chargetransport.

[0015] Other studies have reported electron transfer through DNA usingnonintercalating ruthenium complexes coordinated directly toamino-modified sugars at the terminal position of oligonucleotides(Meade, 1995). In this system it was suggested that electron transfer isprotein-like. In proteins, where the energetic differences in couplingdepend upon σ-bonded interactions, small energetic differences betweensystems do not cause large differences in electronic coupling. In theDNA double helix however, π-stacking can contribute to electroniccoupling such that small energetic differences could lead to largedifferences in coupling efficiency. Most recently, Lewis and coworkersmeasured rates of photo-oxidation of a guanine base in a DNA hairpin byan associated stilbene bound at the top of the hairpin (Lewis, 1997). Bysystematically varying the position of the guanine base within thehairpin and measuring the rate of electron transfer, a value for β, theelectronic coupling parameter, could be made. Here, β was found to beintermediate between that seen in proteins, with σ bonded arrays, andthat found for a highly coupled π-bonded array.

[0016] Electrochemical studies of small molecule/DNA complexes havefocused primarily on solution-phase phenomena, in which DNA-inducedchanges in redox potentials and/or diffusion constants of organic andinorganic species have been analyzed to yield association constants(Carter, 1989, 1990; Rodriguez, 1990; Welch, 1995; Kelly, 1986;Molinier-Jumel, 1978; Berg, 1981; Plambeck, 1984). In addition, rates ofguanine oxidation catalyzed by electrochemically oxidizedtransition-metal complexes have been used to evaluate the solventaccessibility of bases for the detection of mismatches in solution(Johnston, 1995). Electrochemical signals triggered by the associationof small molecules with DNA have also been applied in the design ofother novel biosensors. Toward this end, oligonucleotides have beenimmobilized on electrode surfaces by a variety of linkages for use inhybridization assays. These include thiols on gold (Hashimoto, 1994a,1994b; Okahata, 1992), carbodiimide coupling of guanine residues onglassy carbon (Millan, 1993), and alkane bisphosphonate films onAl³⁺-treated gold (Xu, 1994, 1995). Both direct changes in mass(measured at a quartz crystal microbalance) (Okahata, 1992) and changesin current (Hashimoto, 1994a, 1994b; Millan, 1993) or electrogeneratedchemiluminesence (Xu, 1994, 1995) due to duplex-binding molecules havebeen used as reporters for double stranded DNA. Gold surfaces modifiedwith thiolated polynucleoti des have also been used for the detection ofmetal ions and DNA-binding drugs (Maeda, 1992, 1994).

[0017] Other known electrochemical sensors used in an increasing numberof clinical, environmental, agricultural and biotechnologicalapplications include enzyme based biosensors. Amperometric enzymeelectrodes typically require some form of electrical communicationbetween the electrode and the active site of the redox enzyme that isreduced or oxidized by the substrate. In one type of enzyme electrode, anon-natural redox couple mediates electron transfer from thesubstrate-reduced enzyme to the electrode. In this scheme, the enzyme isreduced by its natural substrate at a given rate; the reduced enzyme isin turn, rapidly oxidized by a non-natural oxidizing component of aredox couple that diffuses into the enzyme, is reduced, diffuses out andeventually diffuses to an electrode where it is oxidized.

[0018] Electrons from a substrate-reduced enzyme will be transferredeither to the enzyme's natural re-oxidizer or, via the redox-centers ofthe polymer to the electrode. Only the latter process contributes to thecurrent. Thus, it is desirable to make the latter process fast relativeto the first. This can be accomplished by (a) increasing theconcentration of the redox centers, or (b) assuring that these centersare fast, i.e. that they are rapidly oxidized and reduced.

[0019] Most natural enzymes are not directly oxidized at electrodes butrequire a mediator either bound to the electrode or in solution. It has,however, been shown that enzymes can be chemically modified by bindingto their proteins redox couples, whereupon, if in the reduced state,they transfer electrons to an electrode. It has also been shown thatwhen redox polycations in solution electrostatically complex polyanionicenzymes, electrons will flow in these complexes from the substrate tothe enzyme, and from the enzyme through the redox polymer, to anelectrode. In addition, systems have been developed where a redox-activepolymer, such as poly(vinyl-pyridine), has been introduced whichelectrically connects the enzyme to the electrode. In this case, thepolycationic redox polymer forms electrostatic complexes with thepolyanionic glucose oxidase in a manner mimicking the natural attractionof some redox proteins for enzymes,, e.g., cytochrome c for cytochrome coxidase.

[0020] The present invention provides a new approach for the detectionof mismatches and common DNA lesions by DNA-mediated charge transport.This electrochemical method is based on DNA-mediated electron transferusing intercalative, redox-active species and detects differences inelectrical current or charge generated with fully base-paired duplexesversus duplexes containing a base-stacking perturbation, such as amismatch. Carried out at an addressable multielectrode array, thismethod allows the processing of multiple sequences in the course of asingle measurement, thus significantly improving the efficiency ofscreening for multiple genetic defects, including detection of only asmall number of mutated DNA samples within a large pool of wild-typesequences and detection of single-base lesions in RNA/DNA hybrids andduplex DNA. Most importantly, the assay reports directly on thestructural difference in base pair stacking within the hybridizedduplex, rather than on a thermodynamic difference based on thecondition-dependent hybridization event itself. Consequently, mismatchdetection becomes independent of the sequence composition and sensorsbased on this approach offer fundamental advantages in scope,sensitivity and accuracy over any other existing methods.

SUMMARY OF THE INVENTION

[0021] The present invention provides a highly sensitive and accuratemethod for the detection of genetic point mutations and common DNAlesions in nucleic acid sequences and its application as a biosensor byDNA-mediated charge transport. The invention also provides anelectrochemical assay of protein binding to DNA-modified electrodesbased upon the detection of associated perturbations in DNA basestacking using DNA charge transport. In a particular embodiment, theinvention relates to electrodes that are prepared by modifying theirsurfaces with oligonucleotide duplexes combined with an intercalative,redox-active species and their use as sensors based on anelectrochemical process in which electrons are transferred between theelectrode and the redox-active species. In another embodiment, theinvention also relates to detection of base-pair mismatches, baseflipping, or other base-stacking perturbations due to protein-nucleicacid interactions. Additionally, the protein-DNA interaction allows amethod of electrical monitoring of DNA enzymatic reactions in a duplexnucleic acid sequence.

[0022] One aspect of the invention relates to methods for determiningthe presence of point mutations sequentially in a series ofoligonucleotide duplexes using an intercalative, redox-active moiety. Apreferred method includes: (a) contacting at least one strand of a firstnucleic acid molecule with a strand of a second nucleic acid moleculeunder hybridizing conditions, wherein one of the nucleic acid moleculesis derivatized with a fanctionalized linker, (b) depositing this duplexonto an electrode or an addressable multielectrode array, (c) contactingthe adsorbed duplex which potentially contains a base-pair mismatch withan intercalative, redox-active moiety under conditions suitable to allowcomplex formation, (d) measuring the amount of electrical current orcharge generated as an indication of the presence of a base-pairmismatch within the adsorbed duplex, (e) treating the complex underdenaturing conditions in order to separate the complex, yielding amonolayer of single-stranded oligonucleotides, and (f) rehybridizing thesingle-stranded oligonucleotides with another target sequence. Steps (c)through (f) can then be repeated for a sequential analysis of variousoligonucleotide probes. Attenuated signals, as compared to the observedsignals for fully base-paired, i.e. wild-type, sequences, willcorrespond to mutated sequences.

[0023] In some instances, it may be desirable to crosslink theintercalative, redox-active species to the duplex and perform the assaycomprised of steps (a) through (d) only.

[0024] Another preferred method relates to the detection of pointmutations utilizing electrocatalytic principles. More specifically, thismethod utilizes an electrode-bound double-stranded DNA monolayer whichis immersed in a solution comprising an intercalative, redox-activespecies, which binds to the monolayer, and a non-intercalativeredox-active species which remains in solution. This method includes:(a) contacting at least one strand of a first nucleic acid molecule witha strand of a second nucleic acid molecule under hybridizing conditions,wherein one of the nucleic acid molecules is derivatized with afunctionalized linker, (b) depositing this duplex which potentiallycontains a base-pair mismatch onto an electrode or an addressablemnultielectrode array, (c) immersing this complex in an aqueous solutioncomprising an intercalative, redox-active moiety and anon-intercalative, redox-active moiety under conditions suitable toallow complex formation, (d) measuring the amount of electrical currentor charge generated as an indication of the presence of a base-pairmismatch within the adsorbed duplex, (e) treating the complex underdenaturing conditions in order to separate the complex, yielding amonolayer of single-stranded oligonucleotides, and (f) rehybridizing thesingle-stranded oligonucleotides with another target sequence. Steps (c)through (f) can then be repeated for a sequential analysis of variousoligonucleotide probes. Utilizing this method, pronounced currents andthus increased signals will be observed due to the electrocatalyticreduction of the non-intercalative, redox-active moiety by thesurface-bound, redox-active moiety.

[0025] In yet another embodiment, the invention provides anelectrocatalytic assay that enables the detection of all possible singlebase mismatches, a small number of mutated DNA samples within a largepool of wild-type sequences, several naturally occurring base lesionsand single base lesions in either DNA or RNA/DNA hybrids. Specifically,this method utilizes an electrode-bound double-stranded DNA monolayerwhich is immersed in a solution comprising an intercalative,redox-active species, which binds to the monolayer surface, and anon-intercalative redox-active species which remains in solution. Thismethod includes, contacting a first single stranded nucleic acid probesequence, which may be derivatized, with a second single strandednucleic acid target sequence to form a duplex, wherein the second singlestranded nucleic acid target sequence is hybridized to a monolayer offirst single stranded nucleic acid probe sequences on an electrode;wherein the monolayer is prepared by first attaching a duplex comprisingthe first single stranded nucleic acid probe sequence to the electrode,dehybridizing the duplex, such that the first single stranded nucleicacid probe sequence remains attached to the electrode, and wherein thehybrid of the first single stranded nucleic acid probe sequence and thesecond single stranded target sequence form a double stranded nucleicacid-modified film; immersing the nucleic acid-modified film in asolution comprising an intercalative, redox-active moiety and anon-intercalative redox-active moiety; and measuring the electricalcurrent or charges of the catalytic reduction of the non-intercalative,redox-active species by the intercalative, redox-active moiety wherein adifference of the electric current or charge from the electrical currentor charge of a nucleic acid duplex not having one or more single-basesequence lesions is indicative of the presence or absence of one or moresingle-base sequence lesions in the target sequence. The last two stepscan be repeated for a sequential analysis of various oligonucleotideprobes. Utilizing this method, pronounced currents and thus increasedsignals will be observed due to the electrocatalytic reduction of thenon-intercalative, redox-active moiety by the surface-bound,redox-active moiety.

[0026] In general, owing to their relatively high thermodynamicstability and their minor modification to the DNA duplex, these lesionshave been very difficult to detect, with postlabeling procedures thepreferred methods of analysis. These lesions do, however, appear toperturb electronic coupling within the DNA duplex. As a result, DNAmediated charge transport coupled to electrocatalysis is sensitive tothose perturbations.

[0027] Yet another aspect of the invention relates to a method ofdetecting the presence or absence of a protein and includes: (a)contacting at least one strand of a first nucleic acid molecule with astrand of a second nucleic acid molecule under hybridizing conditions,wherein one of the nucleic acid molecules is derivatized with afunctionalized linker and wherein the formed duplex is designed such tocontain the recognition site of a nucleic acid-binding protein ofchoice, (b) depositing this duplex onto an electrode or an addressablemultielectrode array, (c) contacting the adsorbed duplex with anintercalative, redox-active moiety under conditions suitable to allowcomplex formation, (d) potentially crosslinking the intercalative,redox-active moiety to the duplex, (e) immersing the complex in a firstsample solution to be analyzed for the presence of the nucleicacid-binding protein, (f) measuring the amount of electrical current orcharge generated as an indication of the presence or absence of thenucleic acid-binding protein in the sample solution, (g) treating thecomplex under appropriate conditions to remove the nucleic acid-bindingprotein, and (h) immersing it in a second sample solution to be analyzedfor the presence of the nucleic acid-binding protein in order toseparate the complex. Steps (e) through (h) can then be repeated for asequential analysis of various sample solutions. Attenuated signals, ascompared to signals measured for a reference solution without thenucleic acid-binding protein, indicate the presence of the nucleicacid-binding protein which is binding to its recognition site, thuscausing a perturbation in base-stacking.

[0028] In another aspect, the invention relates to an electrochemicalassay of protein binding to nucleic acid-modified electrodes, based onthe detection of base flipping in the DNA base stacking using DNA chargetransport and includes: a) at least one strand of a single strandedderivatized nucleic acid sequence containing a protein binding sequenceand an electrochemical probe binding sequence is hybridized to a secondsingle stranded nucleic acid sequence to form a duplex, wherein thesecond single stranded nucleic acid sequence is hybridized to amonolayer of first single stranded nucleic acid sequences on anelectrode. The monolayer is prepared by first attaching a duplexcomprising the first single stranded nucleic acid sequence to theelectrode, and dehybridizing the duplex, such that the first singlestranded nucleic acid sequence remains attached to the electrode, andwherein the hybrid of the first single stranded nucleic acid sequenceand the second single stranded sequence form a double stranded nucleicacid-modified film; b) to this film is added an electrochemical probe,which binds to the electrochemical probe binding sequence of the firstsingle stranded nucleic acid sequence; c) to the double stranded nucleicacid modified film is added a protein, which binds to the proteinbinding sequence of the first single stranded nucleic acid sequence; andd) the charges produced are measured and compared to the chargesmeasured without the protein and a difference of the electrical currentof charge from the electrical current or charge of a nucleic acid duplexnot having a protein bound to the duplex DNA is indicative of thepresence or absence of base flipping due to protein binding in theduplex.

[0029] Another aspect of the present invention relates to anelectrochemical assay of protein binding to nucleic acid-modifiedelectrodes based upon the detection of associated perturbations in theDNA base stacking, using DNA charge transport. The method includes: a)at least one strand of a single stranded derivatized nucleic acidsequence containing a protein binding sequence and an electrochemicalprobe binding sequence is hybridized to a second single stranded nucleicacid sequence to form a duplex, wherein the second single strandednucleic acid sequence is hybridized to a monolayer of first singlestranded nucleic acid sequences on an electrode. Wherein the monolayeris prepared by first attaching a duplex comprising the first singlestranded nucleic acid sequence to the electrode, and dehybridizing theduplex, such that the first single stranded nucleic acidsequence-remains attached to the electrode, and wherein the hybrid ofthe first single stranded nucleic acid sequence and the second singlestranded sequence form a double stranded nucleic acid-modified film; b)to this film is added an electrochemical probe, which binds to theelectrochemical probe binding sequence of the first single strandednucleic acid sequence; c) to the double stranded nucleic acid modifiedfilm is added a protein, which binds to the protein binding sequence ofthe first single stranded nucleic acid sequence; and d) the chargesproduced are measured and compared to the charges measured without theprotein and a difference of the electrical current of charge from theelectrical current or charge of a nucleic acid duplex not having aprotein bound to the duplex DNA is indicative of the presence or absenceof perturbations in the DNA base stacking due to protein binding in theduplex.

[0030] Yet another aspect of the invention relates to a method ofelectrochemical monitoring of DNA enzymatic reactions in a duplexnucleic acid sequence associated with protein binding to the duplexnucleic acid sequence, using DNA charge transport, and includes: a) atleast one strand of a single stranded derivatized nucleic acid sequencecontaining a protein binding sequence and an electrochemical probebinding sequence is hybridized to a second single stranded nucleic acidsequence to form a duplex, wherein the second single stranded nucleicacid sequence is hybridized to a monolayer of first single strandednucleic acid sequences on an electrode. Wherein the monolayer isprepared by first attaching a duplex comprising the first singlestranded nucleic acid sequence to the electrode, and dehybridizing theduplex, such that the first single stranded nucleic acid sequenceremains attached to the electrode, and wherein the hybrid of the firstsingle stranded nucleic acid sequence and the second single strandedsequence form a double stranded nucleic acid-modified film; b) to thisfilm is added an electrochemical probe, which binds to theelectrochemical probe binding sequence of the first single strandednucleic acid sequence; c) to the double stranded nucleic acid modifiedfilm is added a protein, which binds to the protein binding sequence ofthe first single stranded nucleic acid sequence; and d) the chargesproduced are measured and compared to the charges measured without theprotein and a difference of the electrical current of charge from theelectrical current or charge of a nucleic acid duplex not having aprotein bound to the duplex DNA is indicative of the presence or absenceof an enzymatic reaction of the duplex.

[0031] The invention also relates to the nature of the redox-activemoieties. The requirements of a suitable intercalative, redox-activemoiety include the position of its redox potential with respect to thewindow within which the oligonucleotide-surface linkage is stable, aswell as the synthetic feasibility of covalent attachment to theoligonucleotide. In addition, chemical and physical characteristics ofthe redox-active intercalator may promote its intercalation in asite-specific or a non-specific manner. In a preferred embodiment, theredox-active species is in itself an intercalator or a larger entity,such as a nucleic acid-binding protein, that contains an intercalativemoiety.

[0032] The nature of the non-intercalative, redox-active species for theelectrocatalysis based assays depends primarily on the redox potentialof the intercalative, redox-active species utilized in that assay.

[0033] Yet another aspect of the invention relates to the compositionand length of the oligonucleotide probe and methods of generating them.In a preferred embodiment, the probe is comprised of two nucleic acidstrands of equal length. In another preferred embodiment the two nucleicacid strands are of uneven length, generating a single-stranded overhangof desired sequence composition (i.e. a “sticky end”). The length of theoligonucleotide probes range preferably from 12 to 25 nucleotides, whilethe single-stranded overhangs are approximately 5 to 10 nucleotides inlength. These single-stranded overhangs can be used to promotesite-specific adsorption of other oligonucleotides with thecomplementary overhang or of enzymes with the matching recognition site.

[0034] The invention further relates to methods of creating a spatiallyaddressable array of adsorbed duplexes. A preferred method includes (a)generating duplexes of variable sequence composition that arederivatized with a functionalized linker, (b) depositing these duplexeson different sites on the multielectrode array, (c) treating the complexunder denaturing conditions to yield a monolayer of single-strandedoligonucleotides, and (d) hybridizing these single-strandedoliglonucleotides with a complementary target sequence. Anotherpreferred method includes (a) depositing 5 to 10 base-pair longoligonucleotide duplexes that are derivatized on one end with afunctionalized linker and contain single-stranded overhangs(approximately 5 to 10 nucleotides long) of known sequence compositionat the opposite end onto a multielectrode array, and (b) contactingthese electrode-bound duplexes under hybridizing conditions withsingle-stranded or double-stranded oligonucleotides that contain thecomplementary overhang.

[0035] Another aspect of the invention is directed towards the nature ofthe electrode, methods of depositing an oligonucleotide duplex (with orwithout a redox-active moiety adsorbed to it) onto an electrode, and thenature of the linkage connecting the oligonucleotide duplex to theelectrode. In a preferred embodiment, the electrode is gold and theoligonucleotide is attached to the electrode by a sulfur linkage. Inanother preferred embodiment the electrode is carbon and the linkage isa more stable amide bond. In either case, the linker connecting theoligonucleotide to the electrode is preferably comprised of 5 to 20covalent bonds.

[0036] Another aspect of the present invention relates to anelectrochemical assay of protein binding to nucleic acid-modifiedelectrodes based upon the detection of base flipping in the DNA basestacking, using DNA charge transport. This assay provides a method forprobing protein-dependent changes in nucleic acid structure.

[0037] Another aspect of the present invention relates to anelectrochemical assay of protein binding to nucleic acid-modifiedelectrodes based upon the detection of associated perturbations in theDNA base stacking, using DNA charge transport. This assay provides amethod for probing protein-dependent changes in nucleic acid structure.

[0038] Yet another aspect of the invention relates to a method ofelectrochemical monitoring of DNA enzymatic reactions in a duplexnucleic acid sequence associated with protein binding to the duplexnucleic acid sequence, using DNA charge transport. This assay provides amethod for probing protein-dependent changes in nucleic acid structure.

[0039] Yet another aspect of the invention relates to various methods ofdetection of the electrical current or charge generated by theelectrode-bound duplexes combined with an intercalative, redox-activespecies. In a preferred embodiment, the electrical current or charge isdetected using electronic methods, for example voltammetry oramperommetry, or optical methods, for example fluorescence orphosphoresence. In another preferred embodiment, the potential at whichthe electrical current is generated is detected by chronocoulometry.

BRIEF DESCRIPTION OF DRAWINGS

[0040]FIG. 1 is a schematic diagram depicting DNA duplexes used forstudy of distance-dependent reduction of daunomycin. The right insertillustrates the daunomycin-guanine crosslink. The left insert shows thethiol-terminated tether which connects the duplex to the electrodesurface and provides 16 σ-bonds between the electrode and the basestack.

[0041]FIG. 2 illustrates cyclic voltammograms of gold electrodesmodified with daunomycin-crosslinked thiol-terminated duplexes (A)SH-^(5′)ATGGATCTCATCTAC+complement and (B)SH-^(5′)ATCCTACTCATGGAC+complement, where the bold Gs represent thedaunomycin crosslinking site.

[0042]FIG. 3 illustrates cyclic voltammograms of gold electrodesmodified with daunomycin-crosslinked thiol-terminated duplexescontaining TA and CA basepairs. The oligonucleotideSH-^(5′)ATTATATAATTGCT was hybridized with the corresponding complementscontaining either a T or a C opposite from the underlined A.

[0043]FIG. 4 describes the charges (Q_(c)) measured for daunomycin atDNA-modified electrodes containing different single-base mismatches. Toobtain the seven different mismatched duplexes the thiol-modifiedsequence, SH-^(5′)AGTACAGTCATCGCG, was hybridized with the followingseven different complements (the mismatch is indicated in bold, and thespecific basepair and the melting temperature of the duplex is given inparentheses): ^(5′)CGCGATGACTGTACT (TA, T_(m) = 68° C.),^(5′)CGCGACGACTGTACT (CA, T_(m) = 56° C.), ^(5′)CGCGATGTCTGTACT (TT,T_(m) = 57° C.), ^(5′)CGCGATCACTGTACT (CC, T_(m) = 56° C.),^(5′)CGCGATGGCTGTACT (GT, T_(m) = 62° C.), ^(5′)CGCGATGAATGTACT (GA,T_(m) = 60° C.), ^(5′)CGCGATGCCTGTACT (CT, T_(m) = 58° C.).

[0044]FIG. 5 describes the charge obtained for DNA-modified electrodesin the presence of 1.0 μM daunomycin. the identified duplexes of varyingpercentages of GC content were either fully base-paired or contained asingle CA mismatch. Mismatch detection measuring the electrical currentor charge generated was independent of the sequence composition.

[0045]FIG. 6 describes the charges (Q_(c)) measured during the in situdetection of a CA mismatch. Electrodes were derivatized with thesequence SH-^(5′)AGTACAGTCATCGCG, where either a C or a T wasincorporated into the complement across from the underlined A. Usingcyclic voltarmmetry, the electrochemical response of daunomycinnon-covalently bound to duplex-modified electrodes was measured firstfor the intact TA or CA duplexes (TA vs. CA), secondly (afterdenaturation of the duplex) for the single stranded oligonucleotide(ss), thirdly (after rehybridization with the opposite complement) againfor the duplex (CA vs. TA), and lastly (after repeating the denaturationstep) again for the single-stranded oligonucleotide (ss).

[0046]FIG. 7 represents a schematic illustration of electrocatalyticreduction of ferricyanide Methylene blue (MB⁺) is reducedelectrochemically through the DNA base stack to form leucomethylene blue(LB⁺). Ferricyanide is then reduced by LB⁺, causing the regeneration ofMB⁺ and the observation of catalytic currents.

[0047]FIG. 8 illustrates cyclic voltammograms of gold electrodesmodified with thiol-terminated duplexes containing TA and CA basepairsimmersed in a solution containing 1.0 μM methylene blue and 1.0 mMferricyanide. The oligonucleotide SH-^(5′)AGTACAGTCATCGCG was hybridizedwith the corresponding complements containing either a T or a C oppositefrom the underlined A.

[0048]FIG. 9 illustrates the reduction of ferricyanide in theelectrocatalytic process. Electrons flow from the electrode surface tointercalated MB⁺ in a DNA-mediated reaction. The reduced form of MB⁺,leucomethylene blue (LB⁺), in turn, reduces solution-borne ferricyanide,so that more electrons can flow to MB⁺ and the catalytic cyclecontinues.

[0049]FIG. 10 illustrates the charge over time results ofchronocoulometry at −350 mV of 2.0 mM Fe(CN)63-plus 0.5 mM MB+(pH 7) ata gold electrode modified with the thiol-terminated sequenceSH-5′-AGTACAGTCATCGCG hybridized to a fully base paired complement (TA)and complements that introduce single base mismatches. From the top, thefirst line is the TA fully paired complement, the second and third linesare purine-purine mismatches, the fourth and fifth lines arepurine-pyrimidine mismatches, the sixth line is a purine-purinemismatch, the seventh line is a purine-pyrimidine mismatch and theeighth and ninth lines are pyrimidine-pyrimidine mismatches. Theseexperiments were carried out under completely hybridizing conditions atambient temperatures in 5 mM sodium phosphate, 50 mM NaCl, pH 7.0.Mismatch detection is based on diminished DNA-mediated electron transferefficiency due to the local base stack perturbation caused by amismatch. The discrimination between Watson-Crick and mismatchedsequences increases with sampling time.

[0050]FIG. 11 illustrates the charge (uC) measured over time usingelectrocatalytic chronocoulometry at −350 mV with 0.5 M MB⁺ and 2 mMFe(CN)₆ ³⁻ for the sequence SH-5′-ATGGGCCTCCGGTTC with either a CAmismatch at the boldface C (the common 248 mutation), or a CT mismatchat the underlined C (the common 249 mutation), as distinguished from thecharge over time for the native duplex.

[0051]FIG. 12 illustrates the charge (uC) measured over time usingelectrocatalytic chronocoulometry at −350 mV with 0.5 M MB⁺ and 2 mMFe(CN)₆ ³⁻ for various modifications of the sequenceSH-5′AGTACAGTCATCGCG. The electrodes were modified in separate sampleswith double-stranded DNA sequences containing 8-oxo-adenine, 5,6-dihydrothymine, an abasic site, and deoxyuracil paired with guanine (the resultof cytosine deamination). Each lesion was successfully detected withinduplex DNA.

[0052]FIG. 13 illustrates the charge per area of the electrodes in agold microarray after allowing the charge to accumulate during 5 secondsof electrocatalysis. The strictly proportional relationship between thetotal amount of charge accumulated and the electrode can be seen in theFigure.

[0053]FIG. 14 illustrates the charge over time results ofelectrocatalysis of the monolayer on the electrode, the monolayerhybridized to the well-matched sequence SH-5′-AGTACAGTCATCGCG-3′ and themonolayer hybridized to a complement with a single CA mismatch.

[0054]FIG. 15 illustrates the charge over time results ofchronocoulometry at −350 mV of 2.0 mM Fe(CN)63-plus 0.5 mM MB+ (pH 7) ata gold electrode modified with the thiol-terminated sequenceSH-5′-AGTACAGTCATCGCG hybridized to a fully base paired RNA complement(Ta) and other RNA complements that introduce single base mismatches.These experiments were carried out under completely hybridizingconditions at ambient temperatures in 5 mM sodium phosphate, 50 mM NaCl,pH 7.0. Mismatch detection is also achieved in these DNA/RNA hybrids.

[0055]FIG. 16 illustrates a comparison of mismatch detection byelectrocatalysis with varying catalyst concentrations. In lower MB+concentrations, the electrocatalytic signal is shown to decrease rapidlyin a non-linear fashion, as a function of the percent of CA duplexes inthe film. In higher MB⁺ concentrations, however, a linear responseoccurs.

[0056]FIG. 17 illustrates the fabrication of DNA-modified goldelectrodes for electrochemical analysis of protein binding and reaction.Once the gold electrode modification is complete, the electrode isincubated with approximately 1 μM of a test protein for 20 minutes andthen interrogated by chronocoulometry at −575 mV vs. SCE. If the amountof charge accumulated after five seconds is significantly less than thecontrol without protein, that particular protein disrupts the base stackof DNA upon binding.

[0057]FIG. 18 illustrates charge transport through DNA-modifiedsurfaces, which accurately reflects DNA structure perturbation caused byprotein binding. Chronocoulometry at −575 mV of DM covalentlycrosslinked to thiol-terminated DNA films (pH 7) on gold with andwithout bound protein. For the experiments illustrated in panels A-E,the DNA sequence used is above the graph with the DNA binding siteunderlined and the daunomycin binding site in italics. The control traceof that surface is indicated by “DM” (DM only, without protein). Thetest trace after incubation with protein is indicated by “M.HhaI,” UDG,”or “TBP” (demonstrating inhibition of DNA-mediated CT), “Q237W” or“PvuII” (demonstrating efficient CT), or “BSA” (a protein that does notbind DNA). Panels A and B contain the data obtained with M.HhaI; Panel Ais using the well-matched binding site and panel B is using a bindingsite containing an abasic site, which facilitates base-flipping by theenzyme. Panel C contains the data obtained with UDG, anotherbase-flipping enzyme. Panel D contains the results for electrochemicalstudies using TBP and panel E contains the data in the presence andabsence of PvuII. Panel F is a control experiment where the DNA bindingsite (for PvuII) and protein (M.HhaI wildtype, Q237W) do not match orthe protein used does not bind DNA at all (BSA). All of the proteinswhich are known from crystallographic measurements to kink the DNAresult in an inhibition of the DM chronocoulometry (except in panel B,in which the DNA contains an abasic site, which itself is a base stackperturbation), while proteins that bind to DNA without disrupting thebase stack do not result in significant inhibition. A small drop the inamount of charge accumulated is always observed upon protein binding.This small change in the electrical property of the film seems to beindicative of protein binding. For example, when BSA (which does notbind DNA) is used, the small drop in charge is not observed (panels Aand F). However, when M.HhaI is used on a film without a binding site(panel F) but at a concentration where is should bind to DNAnon-specifically, the charge accumulated is diminished a very smallamount, due to protein binding without base flipping. Thus a smalldiminution of charge upon incubation with a protein that does notdisrupt DNA structure, or a large diminution upon incubation with aprotein that does disrupt the DNA structure is evidence that theelectrochemical measurement is a reflection of protein binding.

[0058]FIG. 19 illustrates charge transport through DNA-modifiedsurfaces, which can be used to follow enzyme kinetics. FIG. 19Aillustrates the inculation of DM-crosslinked, surface-boundoligonucleotide duplexes containing the restriction site for PvuII withthe enzyme. As it cuts, the end of the duplex containing the redox probeis released from the surface (FIG. 19A). The release of the redox proberesults in a decrease in the amount of DM reduction over time (FIG,.19B). The graph in FIG. 19B illustrates the plot of amount of chargeaccumulated at the DNA film as a function of enzyme reaction time. Thesame surface incubated without PvuII shows no decrease in DM reductionover time.

[0059] Table 1 describes the electrochemical detection of single-basemismatches based on cyclic voltammograms measured for 1.0 μM daunomycinnoncovalently bound to duplex-modified electrodes.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The expression “amplification of polynucleotides” includesmethods such as polymerase chain reaction (PCR), ligation amplification(or ligase chain reaction, LCR) and amplification methods based on theuse of Q-beta replicase. These methods are well known and widelypracticed in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202and Innis et al., 1990 (for PCR); and Wu et al., 1989a (for LCR).Reagents and hardware for conducting PCR are commercially available.Primers useful to amplify sequences from a particular gene region arepreferably complementary to, and hybridize specifically to sequences inthe target region or in its flanking regions. Nucleic acid sequencesgenerated by amplification may be sequenced directly. Alternatively theamplified sequence(s) may be cloned prior to sequence analysis. A methodfor the direct cloning and sequence analysis of enzymatically amplifiedgenomic segments has been described by Scharf (1986).

[0061] The term “base-stacking perturbations” refers to any event thatcauses a perturbation in base-stacking such as, for example, a base-pairmismatch, a protein binding to its recognition site, an abasic site, abulge, or any other entities that form oligonucleotide adducts.

[0062] The term “base flipping” refers to the process by which a targetnucleic acid base is flipped out of the double helix, making that baseaccessible for reaction.

[0063] The term “denaturing” refers to the process by which strands ofoligonucleotide duplexes are no longer base-paired by hydrogen bondingand are separated into single-stranded molecules. Methods ccofdenaturation are well known to those skilled in the art and includethermal denaturation and alkaline denaturation.

[0064] The term “electrode” refers to an electric conductor thatconducts a current in and out of an electrically conducting medium. Thetwo electrodes, the anode and the cathode, receive and emit electrons,respectively. An electrode is used generally to describe the conductor.In the present invention, an electrode may also be a microarray,consisting of a number of separately addressable electrodes, or anultramicroelectrode.

[0065] The term “hybridized” refers to two nucleic acid strandsassociated with each other which may or may not be fully base-paired.

[0066] The term “intercalative moieties” refers to planar aromatic orheteroaromatic moieties that are capable of partial insertion andstacking between adjacent base pairs of double-strandedoligonucleotides. These moieties may be small molecules or part of alarger entity, such as a protein. Within the context of this inventionthe intercalative moiety is able to generate a response or mediate acatalytic event.

[0067] The term “lesion” refers to an abnormal change in structure ofDNA or RNA. The lesions may include mutations and mismatches in the DNAor RNA and may be naturally occurring, or non-naturally occurring.

[0068] The term “mismatches” refers to nucleic acid bases withinhybridized duplexes which are not 100% complementary. A mismatchincludes any incorrect pairing between the bases of two nucleotideslocated on complementary strands of DNA that are not the Watson-Crickbase-pairs A:T or G:C. The lack of total homology may be due todeletions, insertions, inversions, substitutions or frameshiftmutations.

[0069] The term “mutation” refers to a sequence rearrangement withinDNA. The most common single base mutations involve substitution of onepurine or pyrimidine for the other (e.g., A for G or C for T or viceversa), a type of mutation referred to as a “transition”. Other lessfrequent mutations include “transversions” in which a purine issubstituted for a pyrimidine, or vice versa, and “insertions” or“deletions”, respectively, where the addition or loss of a small number(1, 2 or 3) of nucleotides arises in one strand of a DNA duplex at somestage of the replication process. Such mutations are also known as“frameshift” mutations in the case of insertion/deletion of one of twonucleotides, due to their effects on translation of the genetic codeinto proteins. Mutations involving larger sequence rearrangement alsomay occur and can be important in medical genetics, but theiroccurrences are relatively rare compared to the classes summarizedabove.

[0070] The term “nucleoside” refers to a nitrogenous heterocyclic baselinked to a pentose sugar, either a ribose, deoxyribose, or derivativesor analogs thereof. The term “nucleotide” relates to a phosphoric acidester of a nucleoside comprising a nitrogenous heterocyclic base, apentose sugar, and one or more phosphate or other backbone forminggroups; it is the monomeric unit of an oligonucleotide. Nucleotide unitsmay include the common bases such as guanine (G), adenine (A), cytosine(C), thymine (T), or derivatives thereof. The pentose sugar may bedeoxyribose, ribose, or groups that substitute therefore.

[0071] The terms “nucleotide analog”, “modified base”, “base analog”, or“modified nucleoside” refer to moieties that function similarly to theirnaturally occurring counterparts but have been structurally modified.

[0072] The terms “oligonucleotide” or “nucleotide sequence” refers to aplurality of joined nucleotide units formed in a specific sequence fromnaturally occurring heterocyclic bases and pentofuranosyl equivalentgroups joined through phosphorodiester or other backbone forming groups.

[0073] The terms “oligonucleotide analogs” or “modifiedoligonucleotides” refer to compositions that function similarly tonatural oligonucleotides but have non-naturally occurring portions.Oligonucleotide analogs or modified oligonucleotides may have alteredsugar moieties, altered bases, both altered sugars and bases or alteredinter-sugar linkages, which are known for use in the art.

[0074] The terms “redox-active moiety” or “redox-active species” refersto a compound that can be oxidized and reduced, i.e. which contains oneor more chemical functions that accept and transfer electrons.

[0075] The term “redox protein” refers to proteins that bind electronsreversibly. The simplest redox proteins, in which no prosthetic group ispresent, are those that use reversible formation of a disulfide bondbetween two cysteine residues, as in thioredoxin. Most redox proteinshowever use prosthetic groups, such as flavins or NAD. Many use theability of iron or copper ions to exist in two different redox states.

[0076] The present invention provides a highly sensitive and accuratemethod based on an electrochemical assay using intercalative,redox-active species to determine the presence and location of a singleor multiple base-pair mismatches. Briefly, the system is comprised of(i) a reagent mixture comprising an electrode-bound oligonucleotideduplex to which an intercalative, redox-active moiety is associated and(ii) means for detecting and quantitating the generated electricalcurrent or charge as an indication for the presence of a fullybase-paired versus a mismatch containing duplex. The present inventionis particularly useful in the diagnosis of genetic diseases that arisefrom point mutations. For example, many cancers can be traced to pointmutations in kinases, growth factors, receptors binding proteins and/ornuclear proteins. Other diseases that arise from genetic disordersinclude cystic fibrosis, Bloom's syndrome, thalassemia and sickle celldisease. In addition, several specific genes associated with cancer,such as DCC, NF-1, RB, p53, erbA and the Wilm's tumor gene, as well asvarious oncogenes, such as abl, erbB, src, sis, ras, fos, myb and mychave already been identified and examined for specific mutations.

[0077] The present invention provides methods for detecting single ormultiple point mutations, wherein the oligonucleotide duplex carryingthe redox-active species is adsorbed and therefore continuously exposedto an electrode whose potential oscillates between a potentialsufficient to effect the reduction of said chemical moiety and apotential sufficient to effect the oxidation of the chemical moiety.This method is preferred over other methods for many reasons. Mostimportantly, this method allows the detection of one or more mismatchespresent within an oligonucleotide duplex based on a difference inelectrical current measured for the mismatch-containing versus the fullybase-paired duplex. Thus the method is based on the differences inbase-stacking of the mismatches and is independent of the sequencecomposition of the hybridized duplex, as opposed to existing methodsthat depend on thermodynamic differences in hybridization. Furthermore,this method is nonhazardous, inexpensive, and can be used in a widevariety of applications, alone or in combination with otherhybridization-dependent methods.

[0078] One particular aspect of the invention relates to the method forsequential detection of mismatches within a number of nucleic acidsamples which includes: at least one strand of a nucleic acid moleculeis hybridized under suitable conditions with a first nucleic acid targetsequence forming a duplex which potentially contains a mismatch, andwherein one of the nucleic acids is derivatized with a functionalizedlinker. This duplex is then deposited onto an electrode or anaddressable multielectrode array forming a monolayer. An intercalative,redox-active species (e.g., daunomycin) is noncovalently adsorbed (orcrosslinked, if desired) onto this molecular lawn, and the electricalcurrent or charge generated is measured as an indication of the presenceof a base pair mismatch within the adsorbed oligonucleotide complex.Subsequent treatment of the duplexes containing the intercalative,redox-active species under denaturing conditions allows separation ofthe complex, yielding a single-stranded monolayer of oligonucleotideswhich can be rehybridized to a second oligonucleotide target sequence.The steps of duplex formation, adsorption of the intercalative,redox-active species, measurement of the electrical current or charge,and denaturation of the complex to regenerate the single-strandedoligonucleotides may be repeated as often as desired to detect in asequential manner genetic point mutations in a variety ofoligonucleotide probes.

[0079] The charges passed at each of the electrodes are measured andcompared to the wild-type, i.e. fully base-paired, sequences. Electrodeswith attenuated signals correspond to mutated sequences, while thosewhich exhibit no change in electrical current or charge are unmutated.

[0080] Another aspect of the invention relates to the method ofdetecting mutations utilizing electrocatalysis. Briefly, themodification of electrode surfaces with oligonucleotide duplexesprovides a medium that is impenetrable by negatively charged species dueto the repulsion by the high negative charge of oligonucleotides.However, electrons can be shuttled through the immobilized duplexes toredox-active intercalators localized on the solvent-exposed periphery ofthe monolayer, which in turn can catalytically reduce these negativelycharged species. More specifically, this electrocatalytic methodincludes: at least one strand of a nucleic acid molecule is hybridizedunder suitable conditions with a first nucleic acid target sequenceforming a duplex which potentially contains a mismatch, and wherein oneof the nucleic acids is derivatized with a functionalized linker. Thisduplex is then deposited onto an electrode or a multielectrode arrayforming a monolayer. The assembly is immersed into an aqueous solutioncontaining both an intercalative,, redox-active species (e.g., methyleneblue) and a non-intercalative, redox-active species (e.g.,ferricyanide). The electrical currents or charges corresponding to thecatalytic reduction of ferricyanide mediated by methylene blue aremeasured for each nucleic acid-modified electrode at the potential ofmethylene blue and compared to those obtained with wild-type, i.e. fullybase-paired sequences. Subsequent treatment of the duplexes underdenaturing conditions allows separation of the complex, yielding asingle-stranded monolayer of oligonucleotides which can be rehybridizedto a second oligonucleotide target sequence. The steps of duplexformation, measurement of the catalytically enhanced electrical currentor charge, and denaturation of the complex to regenerate thesingle-stranded oligonucleotides may be repeated as often as desired todetect in a sequential manner genetic point mutations in a variety ofoligonucleotide probes. This particular method based on electrocatalysisat oligonucleotide-modified surfaces is extremely useful for systemswhere attenuated signals resulting from the presence of mismatches aresmall. The addition of a non-intercalative electron acceptor amplifiesthe signal intensity, and allows more accurate measurements. Thisapproach may be particularly useful to monitor assays based onredox-active proteins which bind to the oligonucleotide-modifiedsurface, but are not easily oxidized or reduced because the redox-activecenter is not intercalating.

[0081] Another aspect of the invention relates to the electrocatalyticmethod for detecting all possible single base mismatches, a small numberof mutated DNA samples within a large pool of wild-type sequences,several naturally occurring base lesions and single base lesions ineither DNA or RNA/DNA hybrids. More specifically, this electrocatalyticmethod includes: a first single stranded nucleic acid probe sequence iscontacted with a second single stranded nucleic acid target sequence inorder to form a duplex. The first single stranded nucleic acid may bederivatized with a thiol-terminated alkyl chain, or other derivative.The second single stranded nucleic acid target sequence is hybridized toa monolayer of first single stranded nucleic acid probe sequences on anelectrode. The monolayer of first single stranded nucleic acid probesequences is prepared by first attaching a duplex containing the firstsingle stranded nucleic acid probe sequence to the electrode,dehybridizing the duplex, such that the first single stranded nucleicacid probe sequence remains attached to the electrode. The hybrid of thefirst single stranded nucleic acid probe sequence and the second singlestranded target sequence form a double stranded nucleic acid-modifiedfilm. The nucleic acid-modified film is immersed in a solutioncomprising an intercalative, redox-active moiety (e.g. methylene blue(MB⁺) and a non-intercalative redox-active moiety (e.g. ferricyanide).In the electrocatalytic process, electrons flow from the electrodesurface to intercalated MB⁺ in a DNA-mediated reaction. The reduced formof MB⁺, leucomethylene blue (LB⁺), in turn reduces solution-borneferricyanide, so that more electrons can flow to MB⁺ and the catalyticcycle continues (FIG. 9). Thus in one experiment the surface-bound DNAis repeatedly interrogated. In double-stranded duplexes containing oneor more mismatches, fewer MB⁺ molecules are electrochemically reduced,so the concentration of active catalyst is greatly lowered and theoverall electrocatalytic response is diminished. As a result, thecatalytic reaction amplifies the absolute signal corresponding to MB⁺ inaddition to enhancing the inhibitory affect associated with a mismatch.Furthermore, due to its catalytic nature, the measured charge increaseswith increased sampling times, and in principle is limited only by theconcentration of ferricyanide in solution. The electrical current orcharges of the catalytic reduction of the non-intercalative,redox-active species by the intercalative, redox-active moiety aremeasured and a difference of the electric current or charge from theelectrical current or charge of a nucleic acid duplex not having one ormore single-base sequence lesions is indicative of the presence orabsence of one or more single-base sequence lesions in the targetsequence.

[0082] The present invention further relates to the nature of theredox-active species. These species have a reduced state in which theycan accept electron(s) and an oxidized state in which they can donateelectron(s). The intercalative, redox-active species that are adsorbedor covalently linked to the oligonucleotide duplex include, but are notlimited to, intercalators and nucleic acid-binding proteins whichcontain a redox-active moiety. Covalent attachment of the intercalative,redox-active moiety to the DNA monolayer is not required because bindingis primarily constrained to the top of individual helices within the DNAfilms. The non-covalently bound dye should be intercalatively stacked.Charge transport through the DNA film to the intercalative, redox-activemoiety begins the catalytic cycle. Furthermore, completion of thecatalytic cycle involves reaction with negatively charged ferricyanide,which is electrostatically repulsed from the interior of the anionic DNAfilm. Thus only the intercalative, redox-active moiety bound near thetop of the film is solution accessible, and can therefore participate inthe catalytic cycle.

[0083] An intercalator useful for the specified electrochemical assaysis an agent or moiety capable of partial insertion between stacked basepairs in the nucleic acid double helix. Examples of well-knownintercalators include, but are not limited to, phenanthridines (e.g.,ethidium), phenothiazines (e.g., methylene blue), phenazines (e.g.,phenazine methosulfate), acridines (e.g., quinacrine), anthraquinones(e.g., daunomycin), and metal complexes containing intercalating ligands(e.g., phi, chrysene, dppz). Some of these intercalators may interactsite-selectively with the oligonucleotide duplex. For example, thechrysene ligand is known to intercalate at the mispaired site of aduplex itself (Jackson, 1997), which can be exploited for selectivelocalization of an intercalator. This can be in particular useful toconstruct a duplex monolayer which contains the intercalative,redox-active species exclusively at its periphery.

[0084] The choice of a protein depends on its adsorption and bindingproperties to biological macromolecules, e.g. nucleic acids, and withnon-biological macromolecules, whether in a homogeneous solution, orwhen immobilized on a surface. By changing the absorption or bindingcharacteristics, selectivity, signal to noise ratio and signal stabilityin these assays can be improved. The charge of a protein affects itsadsorption on surfaces, absorption in films, electrophoretic depositionon electrode surfaces, and interaction with macromolecules. It is,therefore, of importance in diagnostic and analytical systems utilizingproteins to tailor the charge of the protein so as to enhance itsadsorption or its binding to the macromolecule of choice, e.g. thenucleic acid. In other cases, e.g. when the detection assay is usedduring several cycles, it is of equal importance to be able tofacilitate desorption, removal, or stripping of the protein from themacromolecule. These assays require oligonucleotide duplexes that aredesigned such as to allow for site-specific binding of the protein ofchoice, which may require a single-stranded overhang. Once the proteinis adsorbed onto the nucleic acid, electrons are relayed via theoligonucleotide duplex to the electrode.

[0085] The nature of the non-intercalative, redox-active species used ina particular electrocatalytic assay depends primarily on the redoxpotential of the intercalating, redox-active species utilized in thatsame assay. Examples include, but are not limited to, any neutral ornegatively charged probes, for example ferricyanide/ferrocyanide,ferrocene and derivatives thereof (e.g., dimethylaminomethyl-,monocarboxylic acid-, dicarboxylic acid-), hexacyanoruthenate, andhexacyanoosmate.

[0086] In the case of nucleic acid-binding proteins, differences inDNA-mediated electron transfer between the duplex-bound protein and theelectrode allow for the detection of base-pair mismatches, baseflipping, or other base-stacking perturbations. Additionally, theprotein-DNA interaction allows a method of electrical monitoring of DNAenzymatic reactions in a duplex nucleic acid sequence.

[0087] One aspect of the present invention relates to an electrochemicalassay of protein binding to nucleic acid-modified electrodes based uponthe detection of base flipping in the DNA base stacking, using DNAcharge transport. This assay provides a method for probingprotein-dependent changes in nucleic acid structure. At least one strandof a single stranded derivatized nucleic acid sequence containing aprotein binding sequence and an electrochemical probe binding sequenceis hybridized to a second single stranded nucleic acid sequence to forma duplex, wherein the second single stranded nucleic acid sequence ishybridized to a monolayer of first single stranded nucleic acidsequences on an electrode. The monolayer is prepared by first attachinga duplex comprising the first single stranded nucleic acid sequence tothe electrode, and dehybridizing the duplex, such that the first singlestranded nucleic acid sequence remains attached to the electrode, andwherein the hybrid of the first single stranded nucleic acid sequenceand the second single stranded sequence form a double stranded nucleicacid-modified film. To this film is added an electrochemical probe,which binds to the electrochemical probe binding sequence of the firstsingle stranded nucleic acid sequence. A protein is added to the doublestranded nucleic acid modified film, which binds to the protein bindingsequence of the first single stranded nucleic acid sequence. The chargesproduced are measured and compared to the charges measured without theprotein and a difference of the electrical current of charge from theelectrical current or charge of a nucleic acid duplex not having aprotein bound to the duplex DNA is indicative of the presence or absenceof base flipping due to protein binding in the duplex.

[0088] In one preferred embodiment, the electrochemical probe isdaunomycin, which is covalently crosslinked to a guanine residue nearthe duplex terminus. In order to ensure the binding site of the redoxprobe, daunomycin is covalently crosslinked to the top of the DNA film.The covalent adduct is formed by reaction with the exocyclic amine inguanine residues in the presence of formaldehyde. All guanines in theduplex are replaced by inosines (I; guanine without the exocyclic amine)except those at the end of the duplex where the daunomycin is intended.In another embodiment, the double stranded nucleic acid-modified film isformed without the presence of Mg²⁺. After assembly of the duplexes, theremaining exposed surface is filled, or “backfilled,” withmercaptohexanol. In one illustrative example, the protein is themethyltransiferase HhaI (M.HhaI), the mutant M.HhaI Q237W, or uracil-DNAglycosylase(See examples 23-28).

[0089] Another aspect of the present invention relates to anelectrochemical assay of protein binding to nucleic acid-modifiedelectrodes based upon the detection of associated perturbations in theDNA base stacking, using DNA charge transport. This assay provides amethod for probing protein-dependent changes in nucleic acid structure.At least one strand of a single stranded derivatized nucleic acidsequence containing a protein binding sequence and an electrochemicalprobe binding sequence is hybridized to a second single stranded nucleicacid sequence to form a duplex, wherein the second single strandednucleic acid sequence is hybridized to a monolayer of first singlestranded nucleic acid sequences on an electrode. The monolayer isprepared by first attaching a duplex comprising the first singlestranded nucleic acid sequence to the electrode, and dehybridizing theduplex, such that the first single stranded nucleic acid sequenceremains attached to the electrode, and wherein the hybrid of the firstsingle stranded nucleic acid sequence and the second single strandedsequence form a double stranded nucleic acid-modified film. To this filmis added an electrochemical probe, which binds to the electrochemicalprobe binding sequence of the first single stranded nucleic acidsequence. A protein is added to the double stranded nucleic acidmodified film, which binds to the protein binding sequence of the firstsingle stranded nucleic acid sequence. The charges produced are measuredand compared to the charges measured without the protein and adifference of the electrical current of charge from the electricalcurrent or charge of a nucleic acid duplex not having a protein bound tothe duplex DNA is indicative of the presence or absence of perturbationsin the DNA base stacking due to protein binding in the duplex.

[0090] In one embodiment, the electrochemical probe is daunomycin, whichis covalently crosslinked to a guanine residue near the duplex terminus.In order to ensure the binding site of the redox probe, daunomycin iscovalently crosslinked to the top of the DNA film. The covalent adductis formed by reaction with the exocyclic amine in guanine residues inthe presence of formaldehyde. All guanines in the duplex are replaced byinosines (I; guanine without the exocyclic amine) except those at theend of the duplex where the daunomycin is intended. In anotherembodiment, the double stranded nucleic acid-modified film is formedwithout the presence of Mg²⁺. After assembly of the duplexes, theremaining exposed surface is filled with mercaptohexanol. In oneillustrative example, the protein used is TATA-box binding protein (TBP)or R.PvuII (See examples 23-28).

[0091] Yet another aspect of the invention relates to a method ofelectrochemical monitoring of DNA enzymatic reactions in a duplexnucleic acid sequence associated with protein binding to the duplexnucleic acid sequence, using DNA charge transport. This assay provides amethod for probing protein-dependent changes in nucleic acid structure.At least one strand of a single stranded derivatized nucleic acidsequence containing a protein binding sequence and an electrochemicalprobe binding sequence is hybridized to a second single stranded nucleicacid sequence to form a duplex, wherein the second single strandednucleic acid sequence is hybridized to a monolayer of first singlestranded nucleic acid sequences on an electrode. The monolayer isprepared by first attaching a duplex comprising the first singlestranded nucleic acid sequence to the electrode, and dehybridizing theduplex, such that the first single stranded nucleic acid sequenceremains attached to the electrode, and wherein the hybrid of the firstsingle stranded nucleic acid sequence and the second single strandedsequence form a double stranded nucleic acid-modified film. To this filmis added an electrochemical probe, which binds to the electrochemicalprobe binding sequence of the first single stranded nucleic acidsequence. A protein is added to the double stranded nucleic acidmodified film, which binds to the protein binding sequence of the firstsingle stranded nucleic acid sequence. The charges produced are measuredand compared to the charges measured without the protein and adifference of the electrical current of charge from the electricalcurrent or charge of a nucleic acid duplex not having a protein bound tothe duplex DNA is indicative of the presence or absence of an enzymaticreaction of the duplex.

[0092] In one embodiment, the electrochemical probe is daunomycin, whichis covalently crosslinked to a guanine residue near the duplex terminus.In order to ensure the binding site of the redox probe, daunomycin iscovalently crosslinked to the top of the DNA film. The covalent adductis formed by reaction with the exocyclic amine in guanine residues inthe presence of formaldehyde. All guanines in the duplex are replaced byinosines (I; guanine without the exocyclic amine) except those at theend of the duplex where the daunomycin is intended. In anotherembodiment, the double stranded nucleic acid-modified film is formedwithout the presence of Mg²⁺. After assembly of the duplexes, theremaining exposed surface is filled with mercaptohexanol. In one aspect,the protein used is a restriction endonuclease, such as PvuII (Seeexamples 23-28).

[0093] Yet another aspect of the invention relates to a method ofdetecting the presence or absence of a protein inducing base-stackingperturbations in DNA duplexes, this method comprising the followingsteps. At least one strand of a nucleic acid molecule is hybridizedunder suitable conditions with a second strand of nucleic acid moleculeforming a duplex, wherein one of the nucleic acids is derivatized with afunctionalized linker. This duplex is designed such to contain therecognition site of a protein of choice at a distinct site along thatduplex. This duplex is then deposited onto an electrode or anaddressable multielectrode array forming a monolayer and anintercalative, redox-active species is adsorbed onto this molecularlawn. In a preferred embodiment, the intercalative, redox-active speciesis site-specifically localized. In another preferred embodiment, theintercalative, redox-active species is crosslinked to theoligoniucleotide duplex. This formed complex is then exposed to a samplesolution that potentially contains the specific protein and theelectrical current or charge generated is measured as an indication ofthe presence or absence of the protein. Subsequently, the protein isremoved under appropriate conditions to regenerate the duplex containingthe intercalative, redox-active moiety. The steps of duplex formation,adsorption or crosslinking of the intercalative, redox-active species,measurement of the electrical current or charge, and regeneration of theduplex containing the intercalative, redox-active moiety may be repeatedas often as desired to detect in a sequential manner the presence of aspecific protein in multiple sample solutions.

[0094] The charges passed at each of the electrodes are measured andcompared to the charges measured in a reference solution without theprotein. Electrodes with attenuated signals indicate the presence of theprotein in question which is binding to its recognition site, thuscausing a perturbation in base-stacking. Examples of proteins that canbe used for this assay include, but are not limited to, restrictionenzymes, TATA-binding proteins, and base-flipping enzymes (e.g., DNAmethylase).

[0095] The present invention also relates to the choice of nucleic acidprobes. Any nucleic acid, DNA or RNA, can be subjected to this mismatchdetection method, provided that the mismatch(es) to be detected liewithin the region between the attachment site of the intercalative,redox-active moiety and the electrode in order to be able to measure adifference in electrical current. The nucleic acid probes to be comparedmay comprise natural or synthetic sequences encoding up to the entiregenome of an organism. These probes can be obtained from any source, forexample, from plasmids, cloned DNA or RNA, or from natural DNA or RNAfrom any source, including bacteria, yeast, viruses, organelles andhigher organisms such as plants and animals. The samples may beextracted from tissue material or cells, including blood cells,amniocytes, bone marrow cells, cells obtained from a biopsy specimen andthe like, by a variety of techniques as described for example byManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Cold Spring Harbor, N.Y. (1982), incorporated hereinby reference.

[0096] Alternatively, the sequences of choice can also be prepared bywell known synthetic procedures. For standard DNA and RNA synthesismethods, see for example “Synthesis and Applications of DNA and RNA” ed.S. A. Narang, Academic Press, 1987, M. J. Gait, “OligonucleotideSynthesis”, IRL Press, Wash. D.C. U.S.A., 1984, and “Oligonucleotidesand Analogues” ed. F. Eckstein, IRL Press, Wash. D.C. U.S.A., 1991, asincorporated herein by reference. Briefly, oligonucleotides andoligonucleotide analogs may be synthesized, conveniently through solidstate synthesis of known methodology. In a preferred embodiment, themonomeric units are added to a growing oligonucleotide chain which iscovalently immobilized to a solid support. Typically, the firstnucleotide is attached to the support through a cleavable linkage priorto the initiation of synthesis. Step-wise extension of theoligonucleotide chain is normally carried out in the 3′ to 5′ direction.When the synthesis is complete, the polymer is cleaved from the supportby hydrolyzing the linkage mentioned above and the nucleotide originallyattached to the support becomes the 3′ terminus of the resultingoligomer. Nucleic acid synthesizers such as the Applied Biosystems,Incorporated 380B are commercially available and their use is generallyunderstood by persons of ordinary skill in the art as being effective ingenerating nearly any oligonucleotide or oligonucleotide analog ofreasonable length which may be desired. Triester, phosphoramidite, orhydrogen phosphonate coupling chemistries are used with thesesynthesizers to provide the desired oligonucleotides or oligonucleotideanalogs.

[0097] In addition, the invention also relates to nucleic acid probesthat are constructed with a defined sequence comprised of nucleotide andnon-natural nucleotide monomers to restrict the number of binding sitesof the intercalative, redox-active agent to one single site. Forexample, in the case of the redox-active intercalator daunomycin mixednucleotide/non-natural nucleotide oligomers were prepared containing A-Tand/or I-C basepairs and one discrete guanine binding site to whichdaunomycin is crosslinked. The non-natural nucleotides are constructedin a step-wise fashion to produce a mixed nucleotide/non-naturalnucleotide polymer employing one of the current DNA synthesis methodswell known in the art, see for example “Synthesis and Applications ofDNA and RNA” ed. S. A. Narang, Academic Press, 1987, M. J. Gait,“Oligonucleotide Synthesis”, IRL Press, Wash. D.C. U.S.A., 1984, and“Oligonucleotides and Analogues” ed. F. Eckstein, IRL Press, Wash. D.C.U.S.A., 1991.

[0098] Methods and conditions used for contacting the oligonucleotidestrands of two DNAs, two RNAs or one DNA and one RNA molecule underhybridizing conditions are widely known in the art. Suitablehybridization conditions may be routinely determined by optimizationprocedures well known to those skilled in the art to establish protocolsfor use in a laboratory. See e.g., Ausubel et al., Current Protocols inMolecular Biology, Vol. 1-2, John Wiley & Sons (1989); Sambrook et al.,Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold SpringsHarbor Press (1989); and Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring, Harbor Laboratory Cold Spring Harbor,N.Y. (1982), all of which are incorporated by reference herein. Forexample, conditions such as temperature, concentration of components,hybridization and washing times, buffer components, and their pH andionic strength may be varied.

[0099] Another aspect of the invention relates to a surface-modifiedelectrode and its use in a bioelectrochemical process, in whichelectrons are transferred directly between an electrode and anelectroactive biological material which is capable of accepting ordonating one or more electrons. Such a bioelectrochemical process can bein either direction. In particular, the invention provides electrodeshaving their surface modified with oligonucleotide duplexes carrying anintercalative, redox-active moiety. The electrode can be of any materialcompatible with the surface modifier being adsorbed or bound thereon,including, but not limited to noble metals such as gold, silver,platinum, palladium, as well as carbon. The preferred material for theelectrodes are gold and carbon.

[0100] The oligonucleotide duplex can be adsorbed onto the electrode inany convenient way. Preferably, the process of preparing such a modifiedelectrode includes adsorbing the oligonucleotide duplex which isderivatized at the 5′-end with a functionalized linker chains onto theelectrode surface in one monolayer to obtain a uniform lawn. Theselinkers include, but are not limited to, thiol- or amine-terminatedchains. This process is generally understood by persons of ordinaryskill in the art as and is relatively simple, reproducible and caneasily be automated.

[0101] Furthermore, the density and composition of the monolayer issubject to variation depending on the selected assay. Methods ofdetecting single base-pair mismatches using intercalative, redox-activemoieties require a densely packed monolayer to prevent the adsorbedintercalative, redox-active moieties from diffusing into the lawn. Themethod for detecting the presence or absence of a protein requirespreferably an uneven monolayer comprised of duplexes of variable lengthto allow the protein to bind effectively to its recognition site alongthe duplex. Alternatively, the monolayer may be less dense. Less densemonolayers may be prepared by a combination of lowering the ionicstrength of the buffer, decreasing the concentration of the derivatizedoligonucleotides in the solution deposited on the electrode, orshortening the time for deposition of the duplex onto the electrode.

[0102] In addition, the present invention further relates to methods ofcreating a spatially addressable array of adsorbed duplexes. In apreferred embodiment, oligonucleotide duplexes of variable sequencecomposition that are derivatized at the 5′-end with a functionalizedlinker are deposited onto a multielectrode array. Subsequent treatmentof these electrode-bound duplexes under denaturing conditions yields amonolayer of single-stranded oligonucleotides, which can then behybridized with a complementary oligonucleotide probe that potentiallycontains a mismatch. In another preferred embodiment, shortoligonucleotide duplexes (5 to 10 base-pairs in length) that arederivatized on one end with a functionalized linker and containsingle-stranded overhangs (5 to 10 nucleotides in length) of designedsequence composition at the opposite end are deposited onto amultielectrode array to generate a spatially addressable matrix. Theseelectrode-bound duplexes can then be hybridized with single-stranded ordouble-stranded oligonucleotides that contain the complementaryoverhang.

[0103] Solid supports containing immobilized molecules have beenextensively used in research, in clinical analyses and in the commercialproduction of foods and chemicals (see e.g., U.S. Pat. No. 5.153,166;Akashi, 1992). Immobilized nucleic acids are used in hybridizationassays (Lamture, 1994) and immobilized proteins in radioimmuno or ELISAassays (see, U.S. Pat. No. 5,314,830). In addition, enzymes have beenimmobilized to facilitate their separation from product and to allow fortheir efficient and repetitive use. A number of important factors haveto be considered in the development of an effective immobilizationprocedure. First, the procedure must minimize non-specific adsorption ofmolecules. Second, the procedure must maintain the, functional integrityof the immobilized molecules. Third, the stability of the bond betweenthe support and the immobilized molecule must be such to avoid leachingwhich would lead to reduced accuracy and sensitivity. Finally, thecoupling procedure must be efficient enough to result in a support witha high capacity for the target molecules as well as be cost effective.

[0104] Another aspect of the invention relates to measuring theelectrical current as a function of degree of hybridization of theoligonucleotide duplex adsorbed onto the electrode. When theintercalative, redox-active species is exposed to electrochemical orchemical energy, the electrical current may be continuously detectedusing techniques well known in the art. These include, but are notlimited electronic methods, for example voltammetry or amperommetry, oroptical methods, for example fluorescence or phosphoresence.

[0105] Generally, photoluminescence excitation and emission occur withelectromagnetic radiation of between about 200 nanometers and about 900nanometers in wavelength. Likewise, chemiluminescent andelectrochemiluminescent emission generally occur with the emittedelectromagnetic radiation being between about 200 nanometers and about900 nanometers in wavelength. The potential at which the reduction oroxidation of the chemical moiety occurs depends upon its exact chemicalstructure as well as factors such as the pH of the solution and thenature of the electrode used. It is well known how to determine theoptimal emission and excitation wavelengths in a photoluminescent systemand the optimal potential and emission wavelength of anelectrochemiluminescent and chemiluminescent system.

[0106] There are many methods for quantifying the amount of luminescentspecies present. The rate of energy input into the system can provide ameasure of the luminescent species. Suitable measurements include, forexample, measurements of electric current when the luminescent speciesis generated electrochemically, the rate of reductant or oxidantutilization when the luminescent species is generated chemically or theabsorption of electromagnetic energy in photoluminescent techniques. Inaddition, the luminescent species can be detected by measuring theemitted electromagnetic radiation. All of these measurements can be madeeither as continuous, event-based measurements, or as cumulative methodswhich add the signal over a long period of time. Event-basedmeasurements may be carried out with photomultiplier tubes, photodiodesor phototransistors to produce electric currents proportional inmagnitude to the incident light intensity, or by using charge coupledevices. Examples of cumulative methods are the integration ofevent-based data, and the use of photographic film to provide cumulativedata directly.

[0107] The publications and other reference materials referred to hereindescribe the background of the invention and provide additional detailregarding its practice and are hereby incorporated by reference. Forconvenience, the reference materials are referenced and grouped in theappended bibliography.

[0108] The invention will be further described with reference to thefollowing examples; however, it is to be understood that the inventionis not limited to such examples.

EXAMPLES Materials

[0109] Phosphoramidite reagents (including the C₆S-S thiol modifier)were obtained from Glen Research. [γ-³²P]dATP was obtained fromNEN-DuPont. Potassium ferrocyanicle (Fisher) was recrystallized fromaqueous solution prior to use. Daunomycin was obtained from Fluka.

Synthesis of Derivatized Duplexes

[0110] Oligonucleotides immobilized on a controlled pore glass resinwere treated in succession with carbonyldiimidazole and 1,6-diaminohexane (1 g/10 ml dioxane, 30 min/ea.) at the 5′-hydroxyterminus before cleavage from the resin (Wachter, 1986). Afterdeprotection, the free amine was treated with 2-pyridylthiopropionicacid N-succinimide ester to produce a disulfide (Harrison, 1997). Thesequences were purified by reverse-phase HPLC, converted to free thiolsusing dithiothreitol, and repurified before hybridization to theircomplements. Derivatized oligonucleotides were characterized bymass-assisted laser desorption ionization time-of-flight massspectrometry and HPLC retention times. Duplexes were hybridized indeoxygenated 5 mM phosphate/50 mM NaCl (pH 7) by heating to 90° C.followed by slow cooling to room temperature. Unprotected duplexes werestored frozen under argon to prevention oxidation of the thiol.

Atomic Force Microscopy (AFM)

[0111] All AFM images were collected using a MultiMode AFM running onthe NanoScope IIIa controller (Digital Instruments, Santa Barbara,Calif.). A glass AFM chamber (Digital Instruments, Santa Barbara,Calif.) and a fluid volume of approximately 50 microliters were used forthe experiments. Si₃N₄ cantilevers (spring constant, 0.06 N/m) withintegrated, end-mounted oxide-sharpened Si₃N₄ probe tips were used. Theapplied vertical force of the AFM probe during imaging was minimized tobeneath 100 pN. Continually adjusting the cantilever deflection feedbacksetpoint compensated for thermal drifting of the cantilever and aconsistent, minimum force was maintained AFM height calibrations werecarried out on a NIST-traceable 180-nm height standard and thenconfirmed by measuring a single-atom step in the Au gold surface. TheAFM images were recorded in “Height” (or constant force) mode. Holes inthe monolayer used to determine monolayer thicknesses were prepared bydecreasing the scan size to approximately 100-150 nm, increasing thescan rate to 24-30 Hz, and increasing the vertical force by advancingthe setpoint several units. After about one minute, the scan size, scanrate, and setpoint were returned to their previous values, and imagesfeaturing a bare gold square were captured. All images captured forheight-contrast analysis were recorded at minimum vertical tip forces.This was accomplished by decreasing the set-point until the tipdisengaged from the surface, then reintroducing it with the minimumforce required to achieve a stable image. In several cases, the filmheight was also measured in tapping mode, and gave the same result asthe contact-mode experiments.

Electrochemistry

[0112] Cyclic voltammetry (CV) was carried out on 0.02 cm²polycrystalline gold electrodes using a Bioanalytical Systems (BAS)Model CV-50W electrochemical analyzer at 20±2° C. in 100 mM phosphatebuffer (pH 7). A normal three-electrode configuration consisting of amodified gold-disk working electrode, a saturated calomel referenceelectrode (SCE, Fisher Scientific), and a platinum wire auxiliaryelectrode was used. The working compartment of the electrochemical cellwas separated from the reference compartment by a modified Luggincapillary. Potentials are reported versus SCE. Heterogeneouselectron-transfer rates were determined and analyzed by CV (Nahir, 1994;Weber, 1994; Tender, 1994).

Ellipsometry

[0113] Optical ellipsometry (λ=632.8 nm) was carried out on driedsamples at 25° C. using a Gaertner Model L116C ellipsometer.

Example 1 Site-Specific Incorporation of a Redox-Active IntercalatorInto a DNA Duplex.

[0114] The redox-active intercalator daunomycin (DM) (Arcamone, 1981)was incorporated into the DNA duplex to investigate charge transductionthrough these duplexes (FIG. 1). DM undergoes a reversible reduction(Molinier-Jumel, 1978; Berg, 1981) within the potential window of themonolayers (Kelley, 1997a), and covalent adducts of intercalated DMcrosslinked to the 2-amino group of guanine (Leng, 1996) have beencrystallographically characterized within duplex DNA (Wang, 1991). Thus,a series of oligonucleotides primarily containing A-T or inosine (I)-Cpairs were constructed with discrete guanine binding sites to which DMwas crosslinked. Preferably, thiol-terminated duplexes (0.1 mM)containing an adjacent pair of guanines were hybridized, incubated with0.2 % formaldehyde and 0.2 mM DM in 5 mM phosphate, 50 mM NaCl, pH 7 for1 h, and phenol extracted to remove excess DM.

[0115] Moving the guanine site along the duplex resulted in a systematicvariation of the through-helix DM/gold separation, and allowed aninvestigation of the effect of distance on the dynamics of chargetransport through the monolayers (FIG. 1).

Example 2 Characterization of DNA Duplexes Modified with a Redox-ActiveIntercalator.

[0116] Modified duplexes were characterized by mass spectrometry,ultraviolet/visible absorption spectroscopy, and thermal denaturationexperiments, all of which were consistent with a 1:1 DM-duplexstoichiometry. For example, the duplexSH—(CH₂)CONH(CH₂)₆NHCO₂-^(5′)ATCCTACTCATGGAC with its inosine complementmodified with DM was analyzed by MALDI-TOF spectrometry. Mass-to-chargeratios (found/calc) of 5284/(5282) (DM+SH strand), 4541/(4540)(complement), and 4742/(4742) (SH strand) were detected. These valuescorrespond to the calculated masses for fragments expected from thisduplex. UV-visible absorption spectroscopy also revealed a 1:1 duplex/DMstoichiometry based upon comparison of the duplex absorbance at 260 nm(M=14.9×10³M⁻¹ cm⁻¹) and the absorbance of intercalated DM at 480 nm(M=5.1×10³M⁻¹ cm⁻¹). In the presence of 100 mM phosphate, 100 mM MgCI₂,and at pH 7, thermal denaturation studies of 5 DM duplexes (monitored byabsorbance at 260 nm) revealed melting temperatures of 48 and 50° C. forthe native and daunomycin-crosslinked duplexes, respectively. A similarmelting profile was obtained by monitoring hypochromicity at 482 nm forthe DM duplex.

Example 3 Preparation of Gold Electrodes Derivatized with DNA Duplexes.

[0117] Electrodes were conveniently prepared by modifying gold surfaceswith 15 base-pair DNA duplexes derivatized at the 5′ end with athiol-terminated alkane chain. Bulk gold electrodes were polishedsuccessively with 0.3- and 0.5-μM alumina (Buhler), sonicated for 30min, and etched in 1.0 M sulfuric acid. Au(111) surfaces were preparedby vapor deposition onto mica or glass (Widrig, 1991; Zei, 1983).Electrodes were then modified by incubation in 0.1 mM solutions ofderivatized DNA duplexes in 5 mM phosphate/50 mM NaCl (pH 7) for 12 -48h at ambient temperature. Modified electrodes were rinsed in bufferprior to use.

[0118] Before deposition of the duplexes onto the gold surfaces, thepresence of the free thiols was confirmed using a spectroscopic assaybased on dithionitrobenzene (Riddles, 1979). Subsequently, the sampleswere deposited onto the gold surfaces for 12-24 h.

[0119] Electrochemical assays, radioactive tagging experiments, andatomic force microscopy (AFM) (Kelley, 1997a, 1997b) all indicate thatthe oligonucleotides form densely packed monolayers oriented in anupright position with respect to the gold surface.

Example 4 Characterization of Modified DNA Duplexes Monolayers on GoldSurfaces

[0120] The DM-modified duplexes readily formed self-assembled monolayerson gold. AFM studies of modified films reveal densely packed monolayerswith heights greater than 45 Å at open circuit. More specific, AFMstudies were carried out under electrochemical control, and revealedthat the DNA films undergo a potential-dependent change in structure. Atopen circuit, the monolayer film height is 45(3) Å. Based on theanisotropic dimensions of the 15-base pair duplexes (20Å in diameter vs.60 Å in length), this thickness indicates that the helical axis isoriented ˜45″ from the gold surface. At applied voltages negative of thepotential of zero charge, film thickness of ˜60 Å are observed; morepositive potential cause a drop in the film height to a limiting valueof 20 Å at low surface coverages.

[0121] Based on the crossectional area of DNA (˜3 nm²) and thegeometrical area of the gold electrodes (0.02 cm²), the maximum surfacecoverage of DNA was calculated as ˜6×10⁻¹¹ mol/cm². Coulometry atelectrodes modified with duplexes containing crosslinked DM revealed aDM surface coverage of 7.5(7)×10⁻¹¹ mol/cm², indicating that the surfaceis densely packed with the modified duplexes. The DM value appeared toexceed slightly the theoretical Γ for DNA, and likely resulted fromadditional electrode surface roughness.

[0122] To assess routinely the surface coverage of DM-derivatized DNA ongold, the electrochemical response of Fe(CN)₆ ⁴⁻ (2 mM) was monitored.This negatively charged ion is repelled from the modified-electrodesurface by the polyanionic DNA, and exhibits essentially no responsewhen the surface is well covered. While not a direct measure of surfacecoverage, this technique allowed the convenient assay of individualelectrodes for adequate modification.

[0123] Cyclic voltammograms of these surfaces showed the reversiblereduction of DM at −0.65 V versus SCE (Molinier-Jumel, 1978; Berg,1981). These films were extremely stable and exhibited responsescharacteristic of surface-bound species (e.g., linear plots of peakcurrent versus scan rate) (Bard, 1980).

Example 5 Measurement of Electrochemical Response of a Redox-ActiveIntercalator Crosslinked to a Fully Base-Paired DNA Duplex on a GoldSurface.

[0124] Integration of the electrochemical response yielded a surfacecoverage (Γ) of electroactive daunomycin of 7.5(7)×10⁻¹¹ mol/cm², avalue in good agreement with the coverages of 15-base pair duplexespreviously measured via ³²P labeling (Kelley, 1997a). However,significant fluctuations in the surface coverages of DM-modifiedduplexes were observed. Therefore, only electrodes which exhibited bothlarge integrated currents for the reduction of crosslinked DM and anattenuated responses for the oxidation of ferrocyanide in solution werestudied.

[0125] Given the 1:1 stoichiometry of crosslinked DM to DNA, theobserved data indicated that all of the bound DM was electrochemicallyreduced. Doping these films with increasing percentages of DM-freeduplexes resulted in a linear decrease in the observed electrochemicalsignals (as determined from coulometric assays), consistent with each ofthe bound intercalators being electrochemically active.

[0126] Remarkably, efficient reduction of DM was observed regardless ofits position along the 15-base-pair sequence as illustrated in FIG. 2.Based on molecular modeling, the DM/gold separations span ˜25 Å. Thethrough-helix DM-electrode separation is >10 Å for DM bound at the endof the duplex closest to the electrode (FIG. 2A), and the DM-electrodeseparation is >35Å (FIG. 2B) for DM crosslinked to the end of the duplexfarthest from the electrode. The surface coverage of electroactivedaunomycin for these 15 base-pair duplexes as measured by integratingthe currents within the illustrated voltammograms were 0.65×10⁻¹⁰mol/cm² and 0.80×10⁻¹⁰ mol/cm², respectively. The DM:DNA stoichiometryfor these same samples, measured by absorption spectroscopy were 0.9:1and 1.1:1, respectively. Thus, the charge did not depend on distance,but did reflect the yield of crosslinking.

Example 6 Measurement of Electrochemical Response of a Redox-ActiveIntercalator Crosslinked to a Mismatch-Containing DNA Duplex on a GoldSurface.

[0127] Electrochemical responses of a redox-active intercalatorcrosslinked to a mismatch-containing DNA duplex on a gold surface weremeasured to determine whether these observed rates were a result ofdirect contact between the redox-active cofactor and the electrodesurface (which has previously been shown to yield apparentlydistance-independent heterogeneous electron transfer (Feng, 1995,1997)). A single site within the 15-base-pair duplex was mutated toproduce a CA mismatch (known to cause local disruptions in the DNA basestack (Patel, 1984; Aboul-ela, 1985) between the intercalated DM and theelectrode surface. FIG. 3 illustrates that such a simple changevirtually eliminated the electrochemical response.

[0128] The coulometry of DM at electrodes modified with CA-containingduplexes varied to some degree as a function of the surface coverage. Athigh surface coverages (as determined by the ferrocyanide assay),essentially no signal was observed with the mismatched duplexes.However, at more moderate surface coverages, small signals correspondingto the reduction of DM were found. These typically did not exceed 30% ofthe signals found for the TA duplexes. The morphology of partial DNAmonolayers is unknown.

[0129] Significantly, sequences in which the positions of the DM and CAmismatch were reversed (such that the mismatch was located above the DMrelative to the gold) showed no diminution in the electrochemicalresponse. AFM images of the CA-mutated sequences were identical to thoseof the TA analogs (monolayer thicknesses of ˜40Å at open circuit),revealing that the bulk structure of the DNA films was not significantlyaltered by the presence of a mismatch. Moreover, the oxidation offerrocyanide was similarly attenuated at both surfaces. Expected massesfor DM-crosslinked DNA duplexes (accounting for the single base change)were measured by mass spectrometry, and spectrophotometric assaysrevealed that the extent of crosslinking was identical in both fullypaired and mismatched sequences.

[0130] The exquisite sensitivity of the electrochemistry of DM tointervening lesions in the base stack provides therefore the basis foran exceptionally versatile DNA-mismatch sensor.

Example 7 Analysis of the Electrochemical Behavior of Fully Base-Pairedor Mismatch-Containing DNA Duplexes Containing Non-CrosslinkedIntercalators.

[0131] A practical method to detect mismatches utilizes a system basedon non-crosslinked, intercalative, redox-active species. Theelectrochemistry of DM non-covalently intercalated into DNA-modifiedfilms was studied in order to develop a general approach to testheterogeneous sequences that may possess more than one guanine-bindingsite. Coulometric titrations confirmed that DM strongly binds tosurfaces modified with fully base-paired duplexes, and yielded affinityconstants very similar to those determined for homogeneous solutions(Arcamone, 1981; Molinier-Jumal, 1978; Berg, 1981). At bulk DMconcentrations ≧1 μM, the modified electrodes were saturated withintercalator, and hold approximately one intercalator persurface-confined duplex. Furthermore, intercalators non-covalently boundto these films exhibited electrochemical properties quite similar tothose described for crosslinked DM, with the exception that the bindingwas reversible, i.e. in pure buffer solutions, decreasing voltammetricsignals were observed until total dissociation was evident.

[0132] In accord with the studies of covalently bound DM, incorporationof a single CA mismatch into these duplexes dramatically decreased theelectrochemical response (Table 1). The magnitude of this mismatcheffect depended strongly on the location of the CA base step along thesequence: when the mutation was buried deep within the monolayer, themeasured charge drops by a factor-of 3.5(5) (relative to theWatson-Crick duplex), but by only 2.3(4) when it was located near thesolvent-exposed terminus. These observations were consistent with DMoccupying sites near the top of the densely packed monolayer, assuggested in earlier studies of methylene blue bound to these samesurfaces (Kelley, 1997b). The intensity of the electrochemical signalstherefore not only reports the presence of the mismatch but also maydescribe the location of the disruption.

[0133] In addition, lateral charge diffusion within these monolayers wasanalyzed. For example, a series of fully base-paired films (sequence:SH-^(5′)AGTACAGTCATCGCG) doped with increasing fractions ofCA-mismatched helices were prepared (the mismatch was localized at thebase step denoted by the bold C in the above sequence.) The coulometricresponse of DM non-covalently bound to these surfaces was stronglydependent on the film composition such that the electrochemical signalsdecreased linearly with increasing percentages of mutated duplexes. Asthere is no measurable difference in the affinities of DM toward TA-versus CA-containing films, this linear response indicated that theelectroinactive intercalators (presumably those molecules bound tomutated helices) are not reduced by lateral charge transfer from theelectroactive species. This result further supports a through-helixpathway for charge transduction, as intermolecular interactions betweenintercalators bound to different duplexes in the film evidently do notmediate efficient electron transfer.

Example 8 Analysis of Mutation Dependence of Electrochemical Response.

[0134] To explore the scope of this mismatch detection strategy, thecharge (Q_(c)) for DM at DNA-modified electrodes containing differentsingle-base mismatches was analyzed (FIG. 4). The seven differentmismatched duplexes were obtained by hybridization of the thiol-modifiedsequence, SH-^(5′)AGTACAGTCATCGCG, with the following seven differentcomplements (the mismatch is indicated in bold, and the specificbasepair and the melting temperature of the duplex is given inparentheses): ^(5′)CGCGATGACTGTACT (TA, T_(m)=68° C.),^(5′)CGCGACGACTGTACT (CA, T_(m)=56° C.), ^(5′)CGCGATGTCTGTACT (TT,T_(m)=57° C.), ^(5′)CGCGATCACTGTACT (CC, T_(m)=56° C.),^(5′)CGCGATGGCTGTACT (GT, T_(m)=62° C.), ^(5′)CGCGATGAATGTACT (GA,T_(m)=60° C.), ^(5′)CGCGATGCCTGTACT (CT, T_(m)=58° C.). The charges werethen calculated by integrating background-subtracted cyclicvoltammograms. The obtained values were based on >5 trials, and theresults were comparable for experiments run side-by-side or utilizingdifferent sample preparations. The melting temperatures of the oligomersin solution were measured by monitoring duplex hypochromicity at 260 nmusing samples that contained 10 μM duplex, 100 mM MgCl₂, and 100 mMphosphate at pH 7.

[0135] Coulometric analysis confirmed that the attenuation of thecharacteristic DM response was strongly dependent upon the identity ofthe mutation. In general, pyrimidine-pyrimidine and purine-pyrimidinemismatches caused marked decreases in the electrochemical signals; theone purine-purine pair studied (a GA mismatch, which is notoriouslywell-stacked within duplex DNA (Patel, 1984; Aboul-ela, 1985)) did notshow a measurable effect. Surprisingly, a significant decrease wascaused by a GT pair, which is also not highly disruptive to the helix.This wobble base pair, although thermodynamically stable, appears tomediate electron transfer poorly.

[0136]FIG. 4 illustrates that across a very narrow range of duplexthermal stabilities, large differences in the electrochemical responsewere observed. Overall, the electrochemical properties of filmscontaining the different mismatches correlated with the degree ofdisruption to base stacking with the individual duplexes. These resultsunderscore the sensitivity of this electrochemical assay to basestacking within DNA, and demonstrate the viability of detectingmismatches based upon charge transduction through thin films.

Example 9 Analysis of Sequence Dependence of Mismatch Detection Assay.

[0137] A single CA mismatch was incorporated into three different DNAduplexes to test for the sequence dependence of the assay. The duplexesfeatured varying percentages of GC content, representing a wide range ofduplex stabilities. The melting temperatures for these duplexes, asdetermined by thermal denaturation measurements obtained by monitoringhypochromicity at 260 nm in duplex solutions containing 10 μM duplex,100 mM phosphate, and 100 mM MgCl₂ were: (SH-^(5′)-ATATAATATATGGAT):TA=47° C., CA=32° C.; (SH-5′AGTACACGTCATCGCG): TA=68° C., CA=56° C.;(SH-^(5′)-GGCGCCCGGCGCCGG): GC=82° C., CA=69° C. The charge wasquantitated from integrating background-subtracted cyclic voltammogramsobtained at υ=100 mV/s and was corrected for electrode area. Asillustrated in FIG. 5, the characteristic drop in coulometric signalsfor DNA duplexes containing a single CA mismatch compared to fullybase-paired DNA films was essentially invariant across AT-rich toGC-rich sequences. This sequence-independent response is not achievableusing traditional mismatch detection assays based upon differentialhybridization.

Example 10 Analysis of Electrochemical Response During Repeated Cycles.

[0138] To extend this methodology to single-stranded targets, techniquesfor in situ hybridization were developed. Thiol-modified duplexes weredeposited on the gold surface, heat denatured, thoroughly rinsed, thenrehybridized with the desired target by incubation in ≧50 pmol ofsingle-stranded oligonucleotide. The electrochemical properties of theresulting surfaces were identical to those described above, suggestingthe suitability of this system for genomic testing.

[0139] For example, a 15-base-pair oligonucleotide,^(5′)AGTACAGTCATCGCG, which was derivatized with a thiol-terminatedlinker, was hybridized both to its native complement and to a mutatedcomplement (at the site underlined in the sequence), generating a fullybase-paired duplex and a CA mismatch-containing duplex, respectively(FIG. 6). These duplexes were deposited on separate electrodes and theelectrochemical responses of DM non-covalently bound to these duplexeswere measured using cyclic voltammetry (ν=100 mV/s, 1.0 μM DM). FIG. 6illustrates that DM exhibited electrochemical responses characteristicof fully base-paired and CA-mutated films, respectively. The surfaceswere then denatured by immersing the electrodes in 90° C. pure bufferfor 2 min to yield single-stranded monolayers of identical sequence.Cyclic voltammetry of DM at these electrodes now revealed nearlyidentical responses, with the reduction appearing highly irreversible,broadened, and becoming smaller as a function of increasing scans.Importantly, the electrode that initially possessed the CA mismatchdisplayed a large signal (for the first scan) after denaturation, whilethe reverse was true for the corresponding TA analog. New duplexes wereformed by incubating the electrodes with 100 pmol of the oppositecomplement in the presence of buffered 100 mM MgCl₂ such that thecomplements were traded (TA→CA, CA→TA), and the electrochemistry at theduplex-modified films again showed the characteristic behavior expectedfor fully base-paired and CA-mutated films. Finally, the electrodes wereagain heated to denature the duplexes and quantitation of the responseshowed again the characteristics for single-stranded oligonucleoltides.Thus, electrodes can be cycled through this sequence of eventsrepeatedly, indicating a practical means to detect point mutationswithin natural DNAs.

Example 11 Detection of Genetic Mutations Within a Specific Region ofthe p53 Gene Using Direct Current Measurement of Thiol-Modified Duplexeson Gold Surfaces.

[0140] A specific embodiment utilizes a gold-microelectrode array withapproximately thirty addressable sites. A different 20-base pair duplexderivatized with a hexylthiol linker is attached to each of these sitesby deposition form a concentrated duplex solution overnight. Thesequences are chosen to correspond to the 600-base pair region withinexons 5 through 8 of the p53 gene where most of the cancer-relatedmutations are found. The array is immersed in aqueous solution at 90° C.for 60 seconds to denature the immobilized duplexes and remove thecomplementary strands. The human sample containing the p53 gene isfragmented either before or after amplification. A solution containingthe fragmented genomic single-stranded DNA is deposited on the array forone hour to allow hybridization to occur. Then, in the presence of a 1.0μM DM solution, the charge passed at each of the electrodes is measured,and the response for each sequence is compared to that obtained from thewild-type (i.e. fully base-paired) sequences. Electrodes with attenuatedsignals correspond to mutated subsequences, while those which exhibitedthe expected charge are classified as unmutated.

Example 12 Detection of Mutations Using Electrocatalytic CurrentsGenerated at DNA-Modified Surfaces.

[0141] The signals corresponding to mismatched and fully-pairedsequences can be more highly differentiated by monitoring catalyticcurrents at DNA-modified surfaces. Electrons can be shuttled through theimmobilized duplexes to redox-active intercalators localized on thesolvent-exposed periphery of the monolayer, and then negatively-chargedsolution-borne species (which are electrostatically prohibited from theinterior of the monolayer) are catalytically reduced by theintercalating mediators. Since the catalytic reaction essentiallyamplifies the signal corresponding to the intercalator, the attenuationof this response in the presence of the mismatch is significantly morepronounced. In a specific embodiment, the sequenceSH-^(5′)AGTACAGTCATCGCG was deposited on an electrode both hybridizedwith a fully base-paired complement, and with a complement containing aCA mismatch (the position of the mismatch is denoted in bold). Theseduplexes were immersed in a solution containing 1.0 μM methylene blueand 1 mM ferricyanide. In the presence of either of these reagentsalone, only small direct currents were measured. However, in thepresence of a mixture of the intercalator and the negatively chargedprobe, pronounced currents were measured corresponding to theelectrocatalytic reduction of ferricyanide by methylene blue. The amountof current observed for the TA and CA containing films differdramatically; using electrocatalysis, the mismatched duplex can bedifferentiated from the fully base-paired duplexes by a factor ofapproximately 3. Moreover, as illustrated in FIG. 8, the peak potentialsfor the TA and CA duplexes are significantly separated, allowing thepresence of the mismatch to be detected potentiometrically. Thisapproach therefore represents an extremely sensitive means to detectgenetic mutations electrochemically.

Example 13 Detection of Genetic Mutations Within a Specific Region ofthe p53 Gene Using Electrocatalytic Current Measurement ofThiol-Modified Duplexes on Gold Surfaces.

[0142] Another specific embodiment involves detecting the mutationswithin the p53 gene using electrocatalysis. A different 20-base pairduplex derivatized with a hexylthiol linker is attached to each ofapproximately thirty addressable sites of a gold-microelectrode array bydeposition form a concentrated duplex solution overnight. The sequencesare chosen to correspond to the 600-base pair region within exons 5through 8 of the p53 gene where most of the cancer-related mutations arefound. The array is immersed in aqueous solution at 90° C. for 60seconds to denature the immobilized duplexes and remove thecomplementary strands. The human sample containing the p53 gene isfragmented either before or after amplification. A solution containingthe fragmented genomic single-stranded DNA is deposited on the array forone hour to allow hybridization to occur. The array is rinsed andsubmerged in a solution containing 1.0 μM methylene blue and 1.0 mMferricyanide. The pronounced currents that are observed result from theelectrocatalytic reduction of the solution-borne ferricyanide bymethylene blue adsorbed at the solvent-exposed duplex sites. Thesecatalytic currents are measured for each addressable electrode andcompared with those obtained with the wild-type sequence to detectpotential sites of mutations.

Example 14 Detection of Genetic Mutations Within a Gene of InterestUsing Direct or Electrocatalytic Current Measurement of Amine-ModifiedDuplexes on Carbon Surfaces.

[0143] Another embodiment utilizes a carbon electrode. The electrode isoxidized at +1.5 V (vs. Ag/AgCl) in the presence of K₂Cr₂O₇ and HNO_(3,)and treated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Duplexescorresponding to mutated sequences of a specific gene of interest arederivatized with a hexylamine linker and applied to the electrodesurface. The device is immersed in aqueous solution at 90 ° C for 60seconds to, generate a single-stranded monolayer, and the fragmentedgenomic DNA sample is hybridized to the immobilized probes at roomtemperature for 1 hour. The detection of mutations is accomplished by(i) measuring direct currents in the presence of 1.0 μM daunomycinsolution, or (ii) by measuring catalytic currents in the presence of 1.0μM methylene blue and 1.0 mM ferricyanide. Charges passed at eachelectrode are measured, and the response for each sequence is comparedto that obtained with wild-type, i.e. fully base-paired, sequences.Attenuated signals correspond to mutated subsequences, while those whichexhibit no change in current are classified unmutated.

[0144] Although the invention has been described with reference toparticular applications, the principles involved may be used in otherapplications which will be apparent to those skilled in the art. Theinvention is accordingly to be limited only by the scope of the claimswhich follow.

Example 15 Fabrication of DNA-Modified Films for Electrocatalysis

[0145] DNA-modified electrodes were prepared by derivatizingsingle-stranded oligonucleotides (typically 15-mers) at the 5′ end witha thiol-terminated alkyl chain [SH(CH₂)₂CONH(CH₂)₆NHCO-DNA], hereinreferred to as SH-5′-DNA. The derivatized single-strandedoligonucleotides were hybridized to their unmodified complements. Theionic strength of the resulting duplex solution was increased with 100mM MgCl₂ and a drop of this solution was placed on the gold surface.After 12-24 h, the electrodes were rinsed thoroughly with buffer andused for electrocatalytic studies. Analyses by radioactive labeling andscanning-probe microscopy indicated that the duplexes formed denselypacked monolayers (50 pmol/cm²) oriented in an upright position withrespect to the gold surface.

[0146] Less dense monolayers (˜1 pmol/cm²) were made using the aboveprocess, and of lowering the ionic strength of the buffer, decreasingthe concentration of the SH-5′-DNA in the deposition solution, orshortening the deposition time.

[0147] After self-assembly, the exposed gold surface was passivatedelectrochemically by polymerization of 2-napthol in order to preventdirect association of the intercalative probe molecules with theelectrode surface, so that electron transfer occurred only throughelectrode-bound duplexes.

Example 16 Detection of All Single Base Mismatches By ElectrocatalyticReduction of Methylene Blue

[0148] In separate experiments, all possible single base mismatches wereincorporated into DNA duplexes using SH-5′-AGTACAGTCATCGCG-3′. Electrodesurfaces were modified with these duplexes and were interrogated usingchronocoulometry (applied potential =−350 mV; 0.5 μM methylenie blue(MB⁺); and 2.0 mM Fe(CN)₆ ³⁻). The marked sensitivity of this assay isevidenced by the detection of all mismatches, including purine-purinemismatches, without any manipulation of experimental conditions (FIG.10). Improved signal differentiation is achieved with increasingsampling time, a result that reflects the catalytic nature of thisassay.

[0149] Mismatched duplexes are easily distinguished from canonicalduplex DNA, but the mismatches cannot be readily distinguished from oneanother, as most yield approximately the same electrocatalytic response.Although GA and AA do yield electrocatalytic currents slightly higherthan the other mismatches, these differences are not great in comparisonto the overall attenuation in signal compared to fully matchedsequences. Nonetheless, this methodology provides a clear strategy todetect a GA mismatch. GA mismatches have not been effectively detectedusing traditional methods owing to their high thermodynamic stability.In fact, even the repair machinery of the cell inefficiently recognizesthe GA mismatch.

[0150] Indeed GA mismatches could not be detected significantly by DNAcharge transport without using the electrocatalysis assay. Mismatchesare generally stacked in a DNA duplex but undergo somewhat greaterdynamical motion than well-paired bases. DNA charge transport detection,which depends upon the electronic coupling within the base stack, issensitive to those motions; electrocatalysis allows for theamplification of this sensitivity.

[0151] Therefore, the electrocatalytic detection of mismatches reliesnot upon their thermodynamic stability but instead upon how well orpoorly the mispair is electronically coupled into the base pair stack.This, in turn, affects how efficiently MB⁺ can be reduced to generateactive catalyst. The ability to detect single-base mismatches,irrespective of sequence, underscores the relevance of this method formutational assays and genetic variability (SNPs).

Example 17 Single-Base Mismatch Detection in Sequences From the Humanp53 Gene

[0152] The p53 tumor suppressor gene encodes a multi-functionaltranscription factor that plays a key role in the prevention of humancancer. Although tumor-inducing mutations have been observed at morethan 100 sites in the p53 gene, more than 90% are found in regions thatencode the DNA binding domain (residues 100-293). Of these, 25% occur atfive “hot spots,” in codons 175, 245, 248, 249, and 273. Hot spotmutations are separated into two categories, class I mutations thataffect key residues at the DNA binding surface (e.g. Arg 248 and Arg273) and class II mutations that affect residues responsible for holdingthe protein in a conformation that readily binds DNA (e.g. Arg 175 andArg 249).

[0153] Two of these mutational hot spots, in codons 248 and 249, werechosen for initial examination using electrocatalysis. The mutation incodon 248 is a class I mutation caused by cigarette smoke that resultsin lung cancer and the 249 mutation is a class II mutation caused byAflatoxin B1 exposure that results in liver cancer. The sequenceSH-5′-ATGGGCCTCCGGTTC includes both of these hot spots and wassynthesized with an alkanethiol linker at the 5′ end. In separatesamples, electrodes were modified with duplexes containing a CA mismatchat the boldface C (the common 248 mutation) or a CT mutation at theunderlined C (the common 249 mutation). As evident in FIG. 11, both ofthese mismatches were successfully differentiated from the native duplexwith electrocatalytic chronocoulometry at −350 mV with 0.5 M MB⁺ and 2mM Fe(CN)₆ ³⁻.

[0154] It is therefore possible to detect mutations within naturalsequences of double-stranded DNA using electrocatalysis. These methodsshould be fully transferable to other genes and sequences.

Example 18 Detection of Naturally Occurring DNA Lesions

[0155] The DNA in living cells is subject to spontaneous alteration aswell as reaction with a variety of chemical compounds and physicalagents present in the cell. For example, the deamination of cytosinegives rise to a CG to AT transition. Reactive oxygen species aregenerated through several pathways including mitochondrial leakage andionizing radiation. 8-oxo-adenine is a possible oxidation product of DNAthat results from the addition of a hydroxyl radical to the C8 positionof the purine base. Hydroxy pyrimidines, such as 5,6-dihydroxy thymines,are another example of base damage products formed via the hydroxylradical during the exposure of DNA to ionizing radiation.

[0156] Several of these possible DNA lesions were incorporated into thesequence SH-5′-AGTACAGTCATCGCG. Electrodes were modified (in separatesamples) with double-stranded DNA sequences containing 8-oxo-adenine,5,6-dihydro thymine, an abasic site, and deoxyuracil paired with guanine(the result of cytosine deamination). The films, once formed, were thenexamined by electrocatalysis at −350 mV with 0.5 M MB⁺ and 2 mM Fe(CN)₆³⁻. These lesions appeared to perturb electronic coupling within the DNAduplex. As a result, DNA mediated charge transport coupled toelectrocatalysis was sensitive to those perturbations. Each lesion wassuccessfully detected within the duplex DNA (FIG. 12).

Example 19 Sensitivity and Detection Limit of the ElectrocatalyticProcess With an Electrode Microarray

[0157] An electrocatalysis assay was run with a gold microarray,consisting of 18 separately addressable gold electrodes ranging in sizeon a micron scale. As can be seen in FIG. 13, the total amount of chargeaccumulated after 5 seconds of electrocatalysis was found to be strictlyproportional to the electrode area, down to electrodes as small as 30 μmor less in diameter.

Example 20 Sensitivity and Detection Limit of the ElectrocatalyticProcess With an Ultramicroelectrode

[0158] Mismatch detection with sequences hybridized at the gold surfaceon the ultramicroelectrodes is also possible. The mismatched sequenceafter in situ hybridization was examined. As illustrated in FIG. 14, theelectrodes on the chip were first modified with the well-matchedsequence SH-5′-AGTACAGTCATCGCG and investigated by electrocatalysis.Then they were gently washed with 90° C. buffer for 2 min followed byrinsing and incubation with a complement containing a single mismatch.Once fully hybridized, electrocatalysis was measured again and theaccumulated charge was found to be characteristic of a mismatchedDNA-monolayer.

[0159] The charge accumulated from the single-stranded monolayer on theultramicroelectrodes was also examined. It is evident thatelectrocatalysis on the single-stranded monolayer results in chargessimilar to films with no mismatch. This is attributed to the fact thatin a single-stranded monolayer (formed by denaturation of thedouble-stranded monolayer) the gold surface is more exposed. Thus MB⁺and ferricyanide are allowed access to the electrode for directcatalysis (not through DNA). Furthermore, the line shape for thesingle-stranded monolayer is different from double-stranded monolayers,which is consistent with a different mechanism for electrocatalysis(direct verses through DNA). The observations with single-stranded DNAalso provided a control demonstrating that detection is not a measure ofhybridization. Both double-stranded (with no mismatches) andsingle-stranded films yielded similar responses. In contrast, themismatched complement, although fully hybridized, yielded an attenuatedsignal.

Example 21 Mismatch Detection in DNA/RNA Hybrids

[0160] In assays for interrogating cellular samples, it may bepreferable to test mRNA transcripts rather than genomic DNA. In thisscenario, patient mRNA was hybridized to a chip or electrode modifiedwith single stranded DNA, resulting in DNA/RNA hybrids. The structure ofsuch duplexes is a hybrid between A- and B-form nucleic acid, whichresults in different base stacking than in pure B-form DNA. Thus allsingle base mismatches within DNA/RNA hybrids were examined byelectrocatalysis to ensure sensitivity to base stacking perturbations inthis alternate duplex structure.

[0161] In separate experiments, all possible single-base mismatches wereincorporated into DNA/RNA hybrids formed using SH-5′-AGTACAGTCATCGCG-3′(the surface-bound strand was DNA and the complements were RNA).Electrode surfaces modified with these duplexes were interrogated usingchronocoulometry. As evident in FIG. 15, all possible mutations weredetected readily. In fact, sensitivity to mutations was almost identicalto the DNA/DNA results (FIG. 10). This means that when testing formutations in clinical samples, genomic DNA, cDNA, or MRNA can beutilized, making electrocatalytic assays flexible and practical forroutine use.

Example 22 Detection of Small Percentages of Mismatched Duplexes WithinWild-Type Films

[0162] In order to investigate the ability of the electrocatalysis assayto detect small numbers of mismatched duplexes within a sea of fullyWatson-Crick paired duplexes on a DNA film, monolayers were constructedin which the percentage of helices featuring a CA base step was variedbetween 0 and 100%. As illustrated in FIG. 16, using the appropriateexperimental conditions (e.g., 0.2 μM MB⁺ and 2 mM ferricyanide) theelectrocatalytic signal decreases rapidly in a non-linear fashion as afunction of the % CA duplexes in the film. Consequently, even smallamounts of mismatched duplexes in the monolayer result in a pronounceddecrease in the electrocatalytic response. Alternatively, using higherconcentrations of MB⁺ (e.g., 5 μM) a linear response occurs (FIG. 16).Such conditions may be useful in the detection of heterozygous genetictraits, in which half of the genes in a diploid organism carry thepotentially harmful variation, and in the detection of somatic mutationsas are associated with cancer.

Example 23 Comparison of CT of DNA-bound Electrodes Formed in thePresence and Absence of Mg2+

[0163] DNA-bound electrodes were formed in the presence of and in theabsence of Mg2+. The two types of surfaces were characterized byscanning probe microscopy with chemically modified Si3N4 tips. Surfaceswith bound l5mer oligonucleotide duplexes formed in the presence of Mg2+were found to produce smooth featureless films with a depth of 45 Å.However, images of DNA surfaces formed without Mg2+ were found to have avery different morphology. The images of DNA surfaces formed withoutMg2+ were seen to have less ordered duplexes on the surface and the goldsurface was visible in some areas. It was seen that without Mg2+, theduplexes were therefore significantly more loosely packed.

[0164] Square wave voltammograms obtained for the electrodes formed withand without Mg2+ showed a reversible reduction of DM at −580 mV versusAg, and possessed features characteristic of surface-bound species. DNAadsorbed on Au(111) without Mg2+ in the self-assembly solution wasquantitated in a 32P radioactive labeling experiment. The assay yieldedan average surface coverage of 12 pmol/cm2 after 24 hours ofmodification, corresponding to a fractional coverage of 0.19. Thesevalues reflected a much lower surface coverage than previous studieswith monolayers formed in the presence of high Mg2+ concentrations. Thestoichiometry of crosslinked DM to DNA as being 1:1 was confirmed byUV-Vis spectroscopy and integration of the electrochemical signal. Alldata indicated that the crosslinked DM is electrochemically active.

Example 24 Detection of Base Flipping Due to Binding of Proteins onDM-DNA Modified Surfaces

[0165] Chronocoulometry results for DM-DNA-modified surfaces in theabsence and presence of different DNA-binding proteins are shown in FIG.17. For the well-matched DNA duplex sequences tested, in the absence ofbound protein, chronocoulometry at −575 mV (versus Ag) yielded asubstantial signal upon integration over 5 sec. In the presence of someproteins, however, signal attenuation was observed, and this diminutionin signal depended upon whether the protein structurally perturbed theDNA.

[0166] The protein-dependent changes are illustrated clearly for DNAfilms containing the methyltransferase HhaI (M.HhaI) target sequence inthe presence and absence of M.HhaI. This enzyme catalyzes themethylation of cytosine in the sequence 5′-GCGC-3′, and a M.HhaI-DNAco-crystal structure revealed that M.HhaI flips the cytosine out fromthe duplex and inserts glutamine 237 into the resulting space in thebase stack. An electrode was prepared containing the duplex sequenceSH-5′-AIAIATICICAIATCC(DM)T-3′ (protein binding site in boldface, DMbinding site in italics). The methyl group source cofactor,S-adenosylmethionine, was omitted to test binding but not reaction onthe electrode surface. As is evident in FIG. 18A, with M.HhtI bound tothe DNA-modified electrode, the amount of charge passing through thefilm was greatly diminished. When BSA, a protein that does not bind DNA,was tested, no inhibition of current flow resulted. The diminution incurrent flow with M.HhaI can be understood based upon the interruptionin the base pair stack as a result of base flipping and glutamineinsertion by M.HhaI.

[0167] Also tested was a mutant M.HhaI, Q237W, that inserts instead anaromatic amino acid side chain, tryptophan, into the base pair stackupon base flipping. With binding of this mutant protein, and restorationof the π-stack, current flow was restored. Also, a small drop in chargewas observed in the binding of the mutant enzyme but not with BSA (videinfra).

[0168] These results support previous observations made in solution,where CT was measured through studies of long range oxidative damage onDNA assemblies containing guanine doublets, as sites of oxidation, and aspatially separated rhodium photooxidant. Oxidative damage at the sitedistal to protein binding was substantially diminished in the presenceof native M.HhaI but was restored in the presence of the Q237W mutant.Parallel results were obtained between these experiments irrespective ofwhether the reaction being monitored involved oxidation chemistry, withthe rhodium photooxidant, or reduction chemistry, here with DM.

Example 25 Effects of Proteins on DM-DNA Modified Surfaces, Where theDNA Films Contained an Abasic Site in the Protein Binding Site

[0169] Analogous results were also shown on DNA films containing anabasic site within the protein binding site (5′-GAbGC-3′). M.HhaI wasseen to bind more tightly to the abasic substrate, but because of helixdisruption, the presence of the abasic site without protein led to adiminution in integrated charge compared to the well-matched substrate.As can be seen in FIG. 18B, binding of the mutant protein with insertionof the Trp residue, however, completed the π-stack and restored currentflow. Thus, the efficient DM reduction observed with Q237W cannot beexplained by a loss of protein binding affinity.

[0170] Square wave voltammetry studies also yielded consistent results.Without protein or with Q237W bound to the film, a reversible peak wasobserved at −580 mV, the reduction potential of DM, but there was nopeak with the wild type enzyme. Therefore only the base stack of DNAwithin protein-DNA complexes was able to be sensitively assayed.

Example 26 Detection of Base Flipping Due to Binding of AdditionalProteins on DM-DNA Modified Surfaces

[0171] Several other structurally well characterized protein-DNAcomplexes were also probed. Uracil-DNA glycosylase, is a base flippingenzyme important to base excision repair. The Au electrode surface wasmodified with the sequence SH-5′-AICTIAATCAITCC(DM)T-3′ with a2′-fluoro-uracil, to prevent enzyme turnover, incorporated onto thecomplementary strand opposite the boldface A. After backfilling withmercaptohexanol and incubation with 1 μM UDG, the surface wasinterrogated at −575 mV by chronocoulometry (FIG. 18C). Again, verylittle charge was transported through this protein-DNA complex. Thisresult further supported the structural model, where base flipping byuracil DNA glycosylase perturbs base stacking and hence current flow toDM.

Example 27 Detection of Perturbations to the Base Stack of a DM-DNAModified Duplex

[0172] The assay also provided a measurement of current flow associatedwith perturbations to the base stack, in addition to base flipping. TheTATA-box binding protein (TBP), for example, kinked DNA approximately90° upon binding to its target site, completely disrupting base stackingbut not base pairing. Binding of TBP to its recognition site wasexamined on electrodes modified with the sequenceSH-5′-IAIATATAAAICACC(DM)T-3′ and mercaptohexanol. As evident in FIG.18D, with protein binding (1 μM), the ability to reduce DM throughDNA-mediated CT was significantly diminished.

[0173] DNA binding by the restriction endonuclease PvuII (R.PvuII) at asurface modified SH-5′-TCTTCAIMTIAIACC(DM)T-3′, passivated withmercaptohexanol, and incubated with 1 μM enzyme was also investigated. Amethylated cytosine (M) was incorporated to prevent cleavage of the DNAfilm during the electrochemical investigation. As predicted from thepresence or absence of even this small change in chronocoulometry isindicative of protein binding. This was also seen in M.HhaI binding toan electrode modified with PvuII DNA (FIG. 18F). Here, M.HhaI was boundto the surface but did not base flip because there was no 5′-GCGC-3′site. BSA, on the other hand, did not bind to DNA and thus no drop incharge was observed.

Example 28 Use of CT for Electrical Probes of Enzyme Reaction Kinetics

[0174] It was also tested whether this assay might be amenable toelectrical probes of DNA enzyme reaction kinetics (FIG. 19). Therestriction activity of PvuII (1 μM) was assayed on the Au surfacemodified with SH-5′-TCTTCAICTIAIACC(DM)T-3′, but with the nativenon-methylated restriction site incorporated into the monolayer tomonitor endonuclease activity. As the enzyme cuts the DNA, the DM probeis released from the surface resulting in a diminution in CT in thefilm. Indeed, the amount of charge accumulated at this surface afterfive seconds of chronocoulcmetry decreased with increasing reaction timewhile the amount of DM reduction in an identical surface without proteinremained constant. The decrease in charge is exponential as expected forthe kinetics of PvuII. Furthermore, the electrochemical data agree witha gel based cleavage assay performed under the electrochemicalconditions. DNA-modified films may therefore be employed also inelectrical monitoring of DNA enzymatic reactions.

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[0275] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

What is claimed is:
 1. A method of detecting one or more single-basesequence lesions in a target nucleic acid sequence, comprising: a)contacting a first single stranded nucleic acid probe sequence with asecond single stranded nucleic acid target sequence to form a duplex,wherein the second single stranded nucleic acid target sequence ishybridized to a monolayer of first single stranded nucleic acid probesequences on an electrode, wherein the monolayer is prepared by firstattaching a duplex comprising the first single stranded nucleic acidprobe sequence to the electrode, and dehybridizing the duplex, such thatthe first single stranded nucleic acid probe sequence remains attachedto the electrode, and wherein the hybrid of the first single strandednucleic acid probe sequence and the second single stranded targetsequence form a double stranded nucleic acid-modified film; b) immersingthe nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety and a non-intercalative redox-activemoiety; and c) measuring the electrical current or charges of thecatalytic reduction of the non-intercalative, redox-active species bythe intercalative, redox-active moiety wherein a difference of theelectric current or charge from the electrical current or charge of anucleic acid duplex not having one or more single-base sequence lesionsis indicative of the presence or absence of one or more single-basesequence lesions in the target sequence.
 2. The method of claim 1,wherein the first single stranded nucleic acid probe sequence contains athiol terminated 5′ end.
 3. The method of claim 2, wherein thethiol-terminated 5′ end is a thiol terminated alkyl chain.
 4. The methodof claim 1, wherein the second single stranded nucleic acid targetsequence is DNA.
 5. The method of claim 1, wherein the second singlestranded nucleic acid target sequence is RNA.
 6. The method of claim 1,wherein the intercalative, redox-active moiety is methylene blue.
 7. Themethod of claim 1, wherein the non-intercalative, redox-active moiety isferricyanide.
 8. A method of detecting one or more single-base sequencelesions in a target nucleic acid sequence, comprising: a) contacting afirst single stranded, derivatized nucleic acid probe sequence with asecond single stranded nucleic acid target sequence to form a duplex,wherein the second single stranded nucleic acid target sequence ishybridized to a monolayer of first single stranded derivatized nucleicacid probe sequences on an electrode, wherein the monolayer is preparedby first attaching a duplex comprising the first single stranded,derivatized nucleic acid probe sequence to the electrode, anddehybridizing the duplex, such that the first single stranded,derivatized nucleic acid probe sequence remains attached to theelectrode, and wherein the hybrid of the first single stranded,derivatized nucleic acid probe sequence and the second single strandedtarget sequence form a double stranded nucleic acid-modified film; b)immersing the nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety and a non-intercalative redox-activemoiety; and c) measuring the electrical current or charges of thecatalytic reduction of the non-intercalative, redox-active species bythe intercalative, redox-active moiety wherein a difference of theelectric current or charge to the electrical current or charge of anucleic acid duplex not having one or more single-base sequence lesionsis indicative of the presence or absence of one or more single-basesequence lesions in the target sequence.
 9. The method of claim 8,wherein the derivatized nucleic acid probe sequence is anoligonucleotide with a thiol-terminated alkyl chain on the 5′ end. 10.The method of claim 9, wherein the oligonucleotide is a DNA sequence.11. The method of claim 8, wherein the second single stranded nucleicacid target sequence is DNA.
 12. The method of claim 8, wherein thesecond single stranded nucleic acid target sequence is RNA.
 13. Themethod of claim 8, wherein the electrode is gold.
 14. The method ofclaim 8, wherein the electrode is carbon.
 15. The method of claim 8,wherein a salt is added to the duplex nucleic acid, before the duplexnucleic acid is deposited onto the electrode.
 16. The method of claim15, wherein the salt is MgCl₂.
 17. The method of claim 15, wherein thesalt added has a concentration of greater than 10 mM.
 18. The method ofclaim 17, wherein the salt has a concentration of 100 mM.
 19. The methodof claim 8, wherein the monolayer of first single stranded derivatizednucleic acid probe sequences is from about 35 pmol/cm² to about 60pmol/cm².
 20. The method of claim 19, wherein the monolayer of firstsingle stranded derivatized nucleic acid probe sequences is about 50pmol/cm².
 21. The method of claim 8, wherein the monolayer of firstsingle stranded derivatized nucleic acid probe sequences is from about0.5 pmol/cm² to about 35 pmol/cm².
 22. The method of claim 21, whereinthe monolayer of first single stranded derivatized nucleic acid probesequences is about 1 pmol/cm².
 23. A method of detecting one or moresingle-base sequence lesions in a target nucleic acid sequence,comprising: a) contacting a first single stranded nucleic acid probesequence with a second single stranded nucleic acid target sequence toform a duplex, wherein the second single stranded nucleic acid targetsequence is hybridized to a monolayer of first single stranded nucleicacid probe sequences on an electrode, wherein the monolayer is preparedby first attaching a duplex comprising the first single stranded nucleicacid probe sequence to the electrode, and dehybridizing the duplex, suchthat the first single stranded nucleic acid probe sequence remainsattached to the electrode, and wherein the hybrid of the first singlestranded nucleic acid probe sequence and the second single strandedtarget sequence form a double stranded nucleic acid-modified film; b)immersing the nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety, wherein the intercalative,redox-active moiety is intercalatively stacked and a non-intercalativeredox-active moiety; and c) measuring the electrical current or chargesof the catalytic reduction of the non-intercalative, redox-activespecies by the intercalative, redox-active moiety wherein a differenceof the electric current or charge to the electrical current or charge ofa nucleic acid duplex not having one or more single-base sequencelesions is indicative of the presence or absence of one or moresingle-base sequence lesions in the target sequence.
 24. The method ofclaim 23, wherein the intercalative, redox-active moiety is methyleneblue.
 25. The method of claim 23, wherein the non-intercalative,redox-active moiety is ferricyanide.
 26. The method of claim 23, whereinthe second single stranded nucleic acid target sequence is DNA.
 27. Themethod of claim 23, wherein the second single stranded nucleic acidtarget sequence is RNA.
 28. The method of claim 23, wherein theintercalative, redox-active moiety is not covalently bound to thenucleic acid-modified film.
 29. A method of detecting one or moresingle-base mismatches in a target nucleic acid sequence, comprising: a)contacting a first single stranded, derivatized nucleic acid probesequence with a second single stranded nucleic acid target sequence toform a duplex, wherein the second single stranded nucleic acid targetsequence is hybridized to a monolayer of first single strandedderivatized nucleic acid probe sequences on an electrode, wherein themonolayer is prepared by first attaching a duplex comprising the firstsingle stranded nucleic acid probe sequence to the electrode, anddehybridizing the duplex, such that the first single stranded nucleicacid probe sequence remains attached to the electrode, and wherein thehybrid of the first single stranded nucleic acid probe sequence and thesecond single stranded target sequence form a double stranded nucleicacid-modified film; b) immersing the nucleic acid-modified film in asolution comprising an intercalative, redox-active moiety and anon-intercalative redox-active moiety; and c) measuring the electricalcurrent or charges of the catalytic reduction of the non-intercalative,redox-active species by the intercalative, redox-active moiety wherein adifference of the electric current or charge to the electrical currentor charge of a nucleic acid duplex not having one or more single-basesequence mismatches is indicative of the presence or absence of one ormore single-base sequence mismatches in the target sequence.
 30. Themethod of claim 29, wherein the single-base mismatch is aguanine-adenine pairing.
 31. The method of claim 29, wherein thesingle-base mismatch is an adenine-adenine pairing.
 32. The method ofclaim 29, wherein the single base mismatch is a guanine-guanine pairing,a thymine-thymine pairing, a cytosine-cytosine pairing, aguanine-thymine pairing, a cytosine-thymine pairing, or acytosine-adenine pairing.
 33. The method of claim 29, wherein the firstsingle stranded derivatized nucleic acid probe sequence is anoligonucleotide with a thiol-terminated alkyl chain on the 5′ end. 34.The method of claim 29, wherein the second single stranded nucleic acidtarget sequence is DNA.
 35. The method of claim 29, wherein the secondsingle stranded nucleic acid target sequence is RNA.
 36. A method ofdetecting one or more single-base sequence lesions in a portion of agene sequence, wherein the one or more single-base sequence lesions isassociated with a disease, comprising: a) contacting a first singlestranded nucleic acid probe sequence, comprising a derivatized portionof the sequence of a gene, with a second single stranded nucleic acidtarget sequence comprising a wild type portion of the gene, to form aduplex, wherein the second single stranded nucleic acid target sequenceis hybridized to a monolayer of first single stranded nucleic acid probesequences on an electrode, wherein the monolayer is prepared by firstattaching a duplex comprising the first single stranded nucleic acidprobe sequence to the electrode, and dehybridizing the duplex, such thatthe first single stranded nucleic acid probe sequence remains attachedto the electrode, and wherein the hybrid of the first single strandednucleic acid probe sequence and the second single stranded targetsequence form a double stranded nucleic acid-modified film; b) immersingthe nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety and a non-intercalative redox-activemoiety; and c) measuring the electrical current or charges of thecatalytic reduction of the non-intercalative, redox-active species bythe intercalative, redox-active moiety wherein a difference of theelectric current or charge to the electrical current or charge of anucleic acid duplex not having one or more single-base sequence lesionsis indicative of the presence or absence of one or more single-basesequence lesions in the gene sequence wherein the base stackingperturbation or single-base sequence lesion is associated with adisease.
 37. The method of claim 36, wherein the portion of the genesequence is up to 200 nucleotides in length.
 38. The method of claim 36,wherein the portion of the gene sequence is from 10 to 50 nucleotid.esin length.
 39. The method of claim 38, wherein the portion of the genesequence is from 15 to 35 nucleotides in length.
 40. The method of claim36, wherein the gene is a human p53 gene.
 41. The method of claim 36,wherein the second single stranded nucleic acid target sequence is DNA.42. The method of claim 36, wherein the second single stranded nucleicacid target sequence is RNA.
 43. A method of detecting one or morenaturally occurring DNA lesions in a target nucleic acid sequence,comprising: a) contacting a first single stranded nucleic acid probesequence containing a DNA lesion with a second single stranded nucleicacid target sequence to form a duplex, wherein the second singlestranded nucleic acid target sequence is hybridized to a monolayer offirst single stranded nucleic acid probe sequences on an electrode,wherein the monolayer is prepared by first attaching a duplex comprisingthe first single stranded nucleic acid probe sequence to the electrode,and dehybridizing the duplex, such that the first single strandednucleic acid probe sequence remains attached to the electrode, andwherein the hybrid of the first single stranded nucleic acid probesequence and the second single stranded target sequence form a doublestranded nucleic acid-modified film; b) immersing the nucleicacid-modified film in a solution comprising an intercalative,redox-active moiety and a non-intercalative redox-active moiety; and c)measuring the electrical current or charges of the catalytic reductionof the non-intercalative, redox-active species by the intercalative,redox-active moiety wherein a difference of the electric current orcharge to the electrical current or charge of a nucleic acid duplex nothaving one or more DNA lesions is indicative of the presence or absenceof one or more DNA lesions in the target sequence.
 44. The method ofclaim 43, wherein the DNA lesion is an oxidized nucleotide.
 45. Themethod of claim 44, wherein the oxidized nucleotide is 8-oxo-adenine.46. The method of claim 43, wherein the DNA lesion is a hydroxypyrimidine.
 47. The method of claim 46, wherein the hydroxy pyrimidineis 5,6-dihydro thymine.
 48. The method of claim 43, wherein the DNAlesion is an abasic site.
 49. The method of claim 43, wherein the DNAlesion is deamination of a nucleotide.
 50. The method of claim 49,wherein the deaminated nucleotide is cytosine.
 51. The method of claim43, wherein the DNA lesion is deoxyuracil paired with guanine.
 52. Themethod of claim 43, wherein the second single stranded nucleic acidtarget sequence is DNA.
 53. The method of claim 43, wherein the secondsingle stranded nucleic acid target sequence is RNA.
 54. A method ofdetecting one or more single-base sequence lesions in a target nucleicacid sequence, comprising: a) contacting a first single stranded nucleicacid probe sequence with a second single stranded nucleic acid targetsequence to form a duplex, wherein the second single stranded nucleicacid target sequence is hybridized to a monolayer of first singlestranded nucleic acid probe sequences on an addressable multielectrodearray, wherein the monolayer is prepared by first attaching a duplexcomprising the first single stranded nucleic acid probe sequence to theaddressable multielectrode array, and dehybridizing the duplex, suchthat the first single stranded nucleic acid probe sequence remainsattached to the addressable multielectrode array, and wherein the hybridof the first single stranded nucleic acid probe sequence and the secondsingle stranded target sequence form a double stranded nucleicacid-modified film; b) immersing the nucleic acid-modified film in asolution comprising an intercalative, redox-active moiety and anon-intercalative redox-active moiety; and c) measuring the electricalcurrent or charges of the catalytic reduction of the non-intercalative,redox-active species by the intercalative, redox-active moiety wherein adifference of the electric current or charge from the electrical currentor charge of a nucleic acid duplex not having one or more single-basesequence lesions is indicative of the presence or absence of one or moresingle-base sequence lesions in the target sequence.
 55. The method ofclaim 54, wherein the multielectrode array comprises gold electrodes.56. The method of claim 54, wherein the multielectrode array comprisescarbon electrodes.
 57. The method of claim 54, wherein the electrodes ofthe multielectrode array are 500 μm or less in diameter.
 58. The methodof claim 54, wherein the second single stranded nucleic acid targetsequence is DNA.
 59. The method of claim 54, wherein the second singlestranded nucleic acid target sequence is RNA.
 60. A method of detectingone or more single-base sequence lesions in a target nucleic acidsequence, comprising: a) contacting a first single stranded nucleic acidprobe sequence with a second single stranded nucleic acid targetsequence to form a duplex, wherein the second single stranded nucleicacid target sequence is hybridized to a monolayer of first singlestranded nucleic acid probe sequences on an ultramicroelectrode, whereinthe monolayer is prepared by first attaching a duplex comprising thefirst single stranded nucleic acid probe sequence to theultramicroelectrode, and dehybridizing the duplex, such that the firstsingle stranded nucleic acid probe sequence remains attached to theultramicroelectrode, and wherein the hybrid of the first single strandednucleic acid probe sequence and the second single stranded targetsequence form a double stranded nucleic acid-modified film; b) immersingthe nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety and a non-intercalative redox-activemoiety; and c) measuring the electrical current or charges of thecatalytic reduction of the non-intercalative, redox-active species bythe intercalative, redox-active moiety wherein a difference of theelectric current or charge from the electrical current or charge of anucleic acid duplex not having one or more single-base sequence lesionsis indicative of the presence or absence of one or more single-basesequence lesions in the target sequence.
 61. The method of claim 60,wherein the second single stranded nucleic acid target sequence is DNA.62. The method of claim 60, wherein the second single stranded nucleicacid target sequence is RNA.
 63. A method of detecting one or moresingle-base sequence lesions in a hybrid of DNA and RNA, comprising: a)contacting a first single stranded nucleic acid probe sequence with asecond single stranded nucleic acid target sequence to form a duplex,wherein the second single stranded nucleic acid target sequence ishybridized to a monolayer of first single stranded nucleic acid probesequences on an electrode, wherein the monolayer is prepared by firstattaching a duplex comprising the first single stranded nucleic acidprobe sequence to the electrode, and dehybridizing the duplex, such thatthe first single stranded nucleic acid probe sequence remains attachedto the electrode, and wherein the hybrid of the first single strandednucleic acid probe sequence and the second single stranded targetsequence form a double stranded nucleic acid-modified film; b) immersingthe nucleic acid-modified film in a solution comprising anintercalative, redox-active moiety and a non-intercalative redox-activemoiety; and c) measuring the electrical current or charges of thecatalytic reduction of the non- inter(alative, redox-active species bythe intercalative, redox-active moiety wherein a difference of theelectric current or charge to the electrical current or charge of anucleic acid duplex not having one or more single-base sequence lesionsis indicative of the presence or absence of the one or more single-basesequence lesions in a hybrid of DNA and RNA.
 64. The method of claim 63,wherein the first single stranded nucleic acid probe sequence is DNA andthe second single stranded nucleic acid target sequence is RNA.
 65. Themethod of claim 64, wherein the RNA is mRNA.
 66. The method of claim 64,wherein the DNA is cDNA.
 67. A method of detecting one or moresingle-base sequence lesions in a portion of a derivatized wild typetarget nucleic acid sample, comprising: a) contacting a first singlestranded nucleic acid probe sequence with a second single strandednucleic acid target sequence, comprising a portion of a derivatized wildtype target nucleic acid sample, to form a duplex, wherein the secondsingle stranded nucleic acid target sequence is hybridized to amonolayer of first single stranded nucleic acid probe sequences on anelectrode, wherein the monolayer is prepared by first attaching a duplexcomprising the first single stranded nucleic acid probe sequence to theelectrode, and dehybridizing the duplex, such that the first singlestranded nucleic acid probe sequence remains attached to the electrode,and wherein the hybrid of the first single stranded nucleic acid probesequence and the second single stranded target sequence form a doublestranded nucleic acid-modified film; b) immersing the nucleicacid-modified film in a solution comprising an intercalative,redox-active moiety and a non-intercalative redox-active moiety; and c)measuring the electrical current or charges of the catalytic reductionof the non-intercalative, redox-active species by the intercalative,redox-active moiety wherein a difference of the electric current orcharge to the electrical current or charge of a nucleic acid duplex nothaving one or more single-base sequence lesions is indicative of thepresence or absence of one or more single-base sequence lesions in aportion of the derivatized wild type target nucleic acid sample.
 68. Themethod of claim 67, wherein the second single stranded nucleic acidtarget sequence is DNA.
 69. The method of claim 67, wherein the secondsingle stranded nucleic acid target sequence is RNA.
 70. A method ofdetecting base flipping in a duplex nucleic acid sequence associatedwith protein binding to the duplex nucleic acid sequence, comprising: a)contacting a first single stranded derivatized nucleic acid sequence,wherein the first single stranded nucleic acid sequence contains aprotein binding sequence and an electrochemical probe binding sequence,with a second single stranded nucleic acid sequence to form a duplex,wherein the second single stranded nucleic acid sequence is hybridizedto a monolayer of first single stranded nucleic acid sequences on anelectrode, wherein the monolayer is prepared by first attaching a duplexcomprising the first single stranded nucleic acid sequence to theelectrode, and dehybridizing the duplex, such that the first singlestranded nucleic acid sequence remains attached to the electrode, andwherein the hybrid of the first single stranded nucleic acid sequenceand the second single stranded sequence form a double stranded nucleicacid-modified film; b) adding an electrochemical probe to the doublestranded nucleic acid-modified film, wherein the electrochemical probebinds to the electrochemical probe binding sequence of the first singlestranded nucleic acid sequence; c) adding a protein to the doublestranded nucleic acid modified film, wherein the protein binds to theprotein binding sequence of the first single stranded nucleic acidsequence; and d) measuring the electrical current or charges of thereduction of the electrochemical probe, wherein a difference of theelectrical current of charge from the electrical current or charge of anucleic acid duplex not having a protein bound to the duplex DNA isindicative of the presence or absence of base flipping due to proteinbinding in the duplex.
 71. The method of claim 70, wherein the firstsingle stranded derivatized nucleic acid sequence is an oligronucleotidewith a thiol-terminated alkyl chain on the 5′ end.
 72. The method ofclaim 71, wherein the oligonucleotide is a DNA sequence.
 73. The methodof claim 70, wherein the electrode is gold.
 74. The method of claim 70,wherein Mg²⁺ is added to the duplex nucleic acid before the duplexnucleic acid is deposited onto the electrode.
 75. The method of claim70, wherein Mg²⁺ is not added to the duplex nucleic acid before theduplex nucleic acid is deposited onto the electrode
 76. The method ofclaim 75, wherein the electrode surface is contacted withmercaptohexanol after the duplex nucleic acid is deposited onto theelectrode.
 77. The method of claim 70, wherein the electrochemical probeis daunomycin (DM).
 78. The method of claim 77, wherein the daunomycinis covalently crosslinked to a guanine residue near the duplex terminusof the first single stranded nucleic acid sequence.
 79. The method ofclaim 70, wherein the protein is methyltransferase HhaI (M.HhaI), Q237W,or uracil-DNA glycosylase.
 80. A method of detecting base stackingperturbations in a duplex nucleic acid sequence associated with proteinbinding to the duplex nucleic acid sequence, comprising: a) contacting afirst single stranded derivatized nucleic acid sequence, wherein thefirst single stranded nucleic acid sequence contains a protein bindingsequence and an electrochemical probe binding sequence, with a secondsingle stranded nucleic acid sequence to form a duplex, wherein thesecond single stranded nucleic acid sequence is hybridized to amonolayer of first single stranded nucleic acid sequences on anelectrode, wherein the monolayer is prepared by first attaching a duplexcomprising the first single stranded nucleic acid sequence to theelectrode, and dehybridizing the duplex, such that the first singlestranded nucleic acid sequence remains attached to the electrode, andwherein the hybrid of the first single stranded nucleic acid sequenceand the second single stranded sequence form a double stranded nucleicacid-modified film; b) adding an electrochemical probe to the doublestranded nucleic acid-modified film, wherein the electrochemical probebinds to the electrochemical probe binding sequence of the first singlestranded nucleic acid sequence; c) adding a protein to the doublestranded nucleic acid modified film, wherein the protein binds to theprotein binding sequence of the first single stranded nucleic acidsequence; and d) measuring the electrical current or charges of thereduction of the electrochemical probe, wherein a difference of theelectrical current of charge from the electrical current or charge of anucleic acid duplex not having a protein bound to the duplex DNA isindicative of the presence or absence of one or more base stackingperturbations in the duplex.
 81. The method of claim 80, wherein thefirst single stranded derivatized nucleic acid sequence is anoligonucleotide with a thiol-terminated alkyl chain on the 5′ end. 82.The method of claim 81, wherein the oligonucleotide is a DNA sequence.83. The method of claim 80, wherein the electrode is gold.
 84. Themethod of claim 80, wherein Mg²⁺ is added to the duplex nucleic acidbefore the duplex nucleic acid is deposited onto the electrode
 85. Themethod of claim 80, wherein Mg2+ is not added to the duplex nucleic acidbefore the duplex nucleic acid is deposited onto the electrode
 86. Themethod of claim 85, wherein the electrode surface is backfilled withmercaptohexanol after the duplex nucleic acid is deposited onto theelectrode.
 87. The method of claim 80, wherein the electrochemical probeis daunomycin (DM).
 88. The method of claim 87, wherein the daunomycinis covalently crosslinked to a guanine residue near the duplex terminusof the first single stranded nucleic acid sequence.
 89. The method ofclaim 80, wherein the protein is TATA-box binding protein (TBP) orrestriction endonuclease PvuII (R.PvuII).
 90. A method of electricalmonitoring of DNA enzymatic reactions in a duplex nucleic acid sequenceassociated with protein binding to the duplex nucleic acid sequence,comprising: a) contacting a first single stranded derivatized nucleicacid sequence, wherein the first single stranded nucleic acid sequencecontains a protein binding sequence and an electrochemical probe bindingsequence, with a second single stranded nucleic acid sequence to form aduplex, wherein the second single stranded nucleic acid sequence ishybridized to a monolayer of first single stranded nucleic acidsequences on an electrode, wherein the monolayer is prepared by firstattaching a duplex comprising the first single stranded nucleic acidsequence to the electrode, and dehybridizing the duplex, such that thefirst single stranded nucleic acid sequence remains attached to theelectrode, and wherein the hybrid of the first single stranded nucleicacid sequence and the second single stranded sequence form a doublestranded nucleic acid-modified film; b) adding an electrochemical probeto the double stranded nucleic acid-modified film, wherein theelectrochemical probe binds to the electrochemical probe bindingsequence of the first single stranded nucleic acid sequence; c) adding aprotein to the double stranded nucleic acid modified film, wherein theprotein binds to the protein binding sequence of the first singlestranded nucleic acid sequence; and d) measuring the electrical currentor charges of the reduction of the electrochemical probe, wherein adifference of the electrical current of charge from the electricalcurrent or charge of a nucleic acid duplex not having a protein bound tothe duplex DNA is indicative of the presence or absence of an enzymaticreaction of the duplex.
 91. The method of claim 90, wherein the firstsingle stranded derivatized nucleic acid sequence is an oligonucleotidewith a thiol-terminated alkyl chain on the 5′ end.
 92. The method ofclaim 91, wherein the oligonucleotide is a DNA sequence.
 93. The methodof claim 90, wherein the electrode is gold.
 94. The method of claim 90,wherein Mg²⁺ is added to the duplex nucleic acid before the duplexnucleic acid is deposited onto the electrode.
 95. The method of claim90, wherein Mg²⁺ is not added to the duplex nucleic acid before theduplex nucleic acid is deposited onto the electrode.
 96. The method ofclaim 95, wherein the electrode surface is backfilled withmercaptohexanol after the duplex nucleic acid is deposited onto theelectrode.
 97. The method of claim 90, wherein the electrochemical probeis daunomycin (DM).
 98. The method of claim 97, wherein the daunomycinis covalently crosslinked to a guanine residue near the duplex terminusof the first single stranded nucleic acid sequence.
 99. The method ofclaim 90, wherein the protein is a restriction endonuclease.
 100. Themethod of claim 99, wherein the restriction endonuclease is PvuII.